The Molecular and Behavioral Pharmacology of the Orphanin

Transcription

0031-6997/01/5303-381–415$3.00
PHARMACOLOGICAL REVIEWS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
Pharmacol Rev 53:381–415, 2001
Vol. 53, No. 3
132/928754
Printed in U.S.A
The Molecular and Behavioral
Pharmacology of the Orphanin
FQ/Nociceptin Peptide and Receptor Family
JEFFREY S. MOGIL AND GAVRIL W. PASTERNAK1
Department of Psychology, McGill University, Montreal, Quebec Canada (J.S.M.); and Laboratory of Molecular Neuropharmacology,
Memorial Sloan-Kettering Cancer Center, New York, New York (G.W.P.)
This paper is available online at http://pharmrev.aspetjournals.org
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Molecular biology of the orphanin FQ/nociceptin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Cloning NOP1 and its gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Alternative splicing NOP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Molecular modifications of NOP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Receptor binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. NOP1 receptor heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V. Orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Structure of orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Orphanin FQ/nociceptin analogs and antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Orphanin FQ/nociceptin precursors and their processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VI. Anatomy of orphanin FQ/nociceptin and its receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Range of effects of orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VIII. Effects of orphanin FQ/nociceptin on pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Effects of supraspinally administered orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Effects of spinally administered orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Effects of peripherally administered orphanin FQ/nociceptin . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Reconciling the literature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Noxious stimulus modality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Robustness of various phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Influence of stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Organismic factors: species, strain, and sex differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Dose-dependency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Opioid tone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Effects of other NOP1 receptor agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Phenotypes of knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. Effects of NOP1 down-regulation or blockade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Antisense studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Pharmacological antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
H. Mechanisms of orphanin FQ/nociceptin actions: ubiquitous cellular inhibition as a unifying
hypothesis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IX. Effects of related peptides on pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Nocistatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Orphanin FQ/nociceptin 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X. Involvement of NOP1 in other central nervous system-mediated behaviors. . . . . . . . . . . . . . . . . . .
A. Locomotor activity and reward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Anxiety, fear, and stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Tolerance and dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Learning and memory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1
Address for correspondence: Dr. Gavril W. Pasternak, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue, New York, NY. E-mail: [email protected]
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ORPHANIN FQ/NOCICEPTIN PEPTIDE AND RECEPTOR FAMILY
E. Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XI. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract——The isolation of an opioid receptor-related clone soon led to the isolation and characterization of a new neuropeptide, termed orphanin FQ or
nociceptin (OFQ/N). This heptadecapeptide binds to
the NOP1 (previously termed ORL1) receptor with exceedingly high affinity, but does not interact directly
with classical opioid receptors. Functionally, the actions of OFQ/N are diverse and intriguing. Most work
has focused upon pain mechanisms, where OFQ/N has
potent anti-analgesic actions supraspinally and analgesic actions spinally. Other OFQ/N activities are less
clear. The diversity of responses might reflect NOP1
receptor heterogeneity, but this remains to be established. The actions of this neurochemical system may
also be uniquely dependent on contextual factors,
both genetic and environmental. This review will address the molecular biology and behavioral pharmacology of OFQ/N and its receptor.
I. Introduction
TABLE 1
Initial reports of the cloning of the NOP1 receptor
The use of molecular biological approaches has led to
extraordinary advances in our understanding of opioid
action in the last decade. Soon after the cloning of the ␦
opioid peptide receptor (DOP1, originally termed DOR-1)
(Evans et al., 1992; Kieffer et al., 1992), a number of
laboratories identified clones corresponding to the ␮ opioid peptide (MOP1, originally termed MOR-1) and ␬
opioid peptide (KOP1, originally termed KOR-1) receptors. A meeting held in Washington, DC, as a tribute to
the memory of Dr. William Martin,2 documented these
advances in opioid receptor pharmacology (Uhl et al.,
1994). At this meeting, several laboratories first described a fourth receptor clone closely homologous to the
traditional opioid receptors (Table 1). These clones were
isolated from a number of species and were typical Gprotein-coupled receptors with the expected predicted
seven transmembrane domains. These novel clones displayed approximately 50% identity with the traditional
opioid receptors overall, with the transmembrane regions showing even higher homologies of up to 80%.
Despite their close homology to the other opioid receptors, the novel clones were difficult to characterize and
were considered by many to be an orphan receptor. Few
opioids labeled these novel clones, and their affinities
were markedly lower than those seen with the cloned
opioid receptors. Functionally, the ability of opioids to
modulate adenylate cyclase with these clones also was
markedly limited (Mollereau et al., 1994; Pan et al.,
1995). Several early papers uncovered a close relationship between this clone and the ␬3 receptor but concluded that they were not identical (Pan et al., 1994,
1995). The evidence for a relationship between them
came from several lines of investigation. A monoclonal
antibody capable of neutralizing ␬3 opioid binding in
brain tissue and ␬3 analgesia in vivo recognized the
2
NIDA Technical Review “The Molecular Neurobiology and Pharmacology of Opiate Receptor Subtypes: A Tribute to William Martin”
held in Washington, DC, November 6 –7, 1993.
Species
Nomenclature
Reference
Mouse
KOR-3
MOR-C
LC132
XOR1
Pan et al., 1994, 1995
Nishi et al., 1994
Bunzow et al., 1994
Wang et al., 1994
Wick et al., 1994
Chen et al., 1994
Lachowicz et al., 1995
Fukuda et al., 1994
Keith et al., 1994
Mollereau et al., 1994
Rat
Ratxor1
C3
ROR-C
Human
ORL1
The NOP1 receptor was cloned from several species by a number of groups. A
listing of the initial descriptions and their nomenclature is presented above. Since
these initial reports, a large number of laboratories have been involved with the
study of this receptor.
expressed receptor generated through in vitro translation in Western blot analysis. Furthermore, in antisense
mapping studies a number of antisense probes directed
against the second and third coding exons of the murine
clone (KOR-3) blocked the analgesic activity of ␬3 analgesic naloxone benzoylhydrazone in mice without affecting the analgesic actions of traditional ␮, ␦, and ␬1
drugs. Yet, the inability of a range of antisense probes
targeting the first coding exon and the markedly different binding profile of the new clone indicated that the
novel clone was not identical to the ␬3 receptor. The
differences between the two were further documented
with the identification of the endogenous ligand for this
receptor, a heptadecapeptide termed orphanin FQ or
nociceptin (OFQ/N3) (Fig. 1). Despite an exceedingly
3
Abbreviations: OFQ/N, orphanin FQ/nociceptin; NalBzoH, naloxone benzoylhydrazone; kb, kilobase(s); bp, base pair(s); CHO, Chinese
hamster ovary; ppOFQ/N, prepro-OFQ/N; DAMGO, [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin; DPDPE, [D-Pen2,D-Pen5]-enkephalin; DSLET, [D-Ser2,Leu5]-enkephalin-Thr; PAG, periaqueductal gray; RVM,
rostral ventromedial medulla; GABA, ␥-aminobutyric acid; ACTH, adrenocorticotrophic hormone; CCK, cholecystokinin; MSH, melanocytestimulating hormone; SBL, scratching, biting, and licking; NMDA, Nmethyl-D-aspartate; NK, neurokinin; CNS, central nervous system;
HPA, hypothalamic-pituitary-adrenal; PG, prostaglandin; VTA, ventral
tegmental area; CPP, conditioned place preference; LTP, long-term
potentiation; VMH, ventromedial hypothalamus.
MOGIL AND PASTERNAK
FIG. 1. OFQ/N and its related peptides. Sequences of the endogenous
opioid peptides and ppOFQ/N peptides are presented. In some situations,
sequences are species-dependent. The sequence of ppOFQ/N160 –187 is
from the mouse and nocistatin from human.
high affinity for the cloned receptor, OFQ/N does not
compete binding to the traditional ␮ opioid receptors.
The nomenclature in this field is confusing, particularly for the receptor. In the early papers each laboratory used its own nomenclature for the receptor (Table
1), but as the years went by the term ORL1 gained favor.
Recently, it has been suggested that all the opioid receptor family of receptors be renamed, with the term NOP1
(nociceptin/orphanin FQ peptide) receptor referring to
the ORL1 clone in all species. Similarly, the ligand is
known as nociceptin or orphanin FQ, having been isolated by two groups independently (Meunier et al., 1995;
Reinscheid et al., 1995). Nociceptin was chosen by one
group to denote its presumed pronociceptive activity.
The term orphanin FQ refers to its affinity for the “orphan” opioid receptor, while the F and Q refer to the first
and last amino acids, phenylalanine and glutamine. Neither term predominates and most laboratories use the
two together, as is done in this review: orphanin FQ/
nociceptin, or OFQ/N.
This field has burgeoned enormously since the cloning
of the receptor and the identification of its peptides.
Aspects of this field have been reviewed previously
(Henderson and McKnight, 1997; Meunier, 1997; Civelli
et al., 1998; Darland et al., 1998; Taylor and Dickenson,
1998; Zaki and Evans, 1998; Yamamoto et al., 1999; also
see a special issue of the journal Peptides, volume 21,
number 7, 2000). The current review will focus rather
comprehensively upon the behavioral pharmacology of
OFQ/N, with an attempt to understand it from the molecular perspective.
II. Molecular Biology of the Orphanin
FQ/Nociceptin Receptor
A. Cloning NOP1 and Its Gene
NOP1 was cloned from various species by a number of
laboratories at about the same time. NOP1 is a typical
G-protein-coupled receptor with seven predicted transmembrane domains (Fig. 2A) and is localized to murine
chromosome 2 (Chen et al., 1994; Nishi et al., 1994). It
383
was readily detected by Northern analysis, where a major band of 3.4 kb was detected in mice (Pan et al., 1995).
Several laboratories found a similar band in rats at
approximately 3.4 kb, as well as additional bands (Chen
et al., 1994; Wang et al., 1994; Lachowicz et al., 1995).
One group reported additional bands of 7.5 and 10 kb
(Chen et al., 1994), another observed a single additional
band of approximately 7.6 kb (Lachowicz et al., 1995),
and a third group reported two additional bands of 13
and 23 kb (Wang et al., 1994). The significance of these
additional larger bands is not clear, particularly with
the differences noted among groups. However, the differing ratios of these larger bands to the 3.4-kb band
among regions raising interesting questions regarding
regional processing (Wang et al., 1994; Lachowicz et al.,
1995).
Southern analysis from a number of groups implies a
single copy of the gene for the NOP1 receptor, which is
termed Oprl1. The murine NOP1 gene structure was
elucidated soon after the initial reports of the receptor
FIG. 2. Schematic of the NOP1 receptor and its gene, Oprl1. A, schematic of the NOP1 receptor is presented with the sites of the protein
corresponding to the splice sites in the mRNA indicated. B, schematic of
the structure of the murine Oprl1 gene.
(Pan et al., 1996b). The receptor has three coding exons,
similar to the other opioid receptors. The first coding
exon yields the amino terminus and the first transmembrane domain (Fig. 2A). The second coding exon is responsible for the next three transmembrane domains.
The splice site between the second and third coding
exons is located in the second extracellular loop, and the
third coding exon is responsible for the remainder of the
protein, including the last three transmembrane domains and the intracellular carboxyl tail (Fig. 2A). The
binding pocket has been proposed to comprise several of
the transmembrane domains (Topham et al., 1998;
Mouledous et al., 2000). The initial gene structure identified five exons, with a noncoding exon preceding and
following the three coding exons (Fig. 2B) (Pan et al.,
1996b). More recent work has identified two mini-exons
between the first and second coding exons that are al-
384
ternatively spliced for a total of seven. The numbering of
the exons has changed, as new ones have been uncovered. In the current review, exon 2 corresponds to the
first coding exon of the original clone, exon 5 to the
second coding exon and exon 6 to the last one.
B. Alternatively Splicing NOP1
Like other members of the opioid receptor family,
NOP1 undergoes alternative splicing. The first variant,
NOP1d, was identified in lymphocytes and contains a
15-bp deletion from the 3⬘ end of the first coding exon
(exon 2) (Halford et al., 1995; Wick et al., 1995). Similar
variants were obtained from mouse (Pan et al., 1998b),
rat (Wick et al., 1994; Xie et al., 1999), and human
(Peluso et al., 1998) brains. An intron retention variant,
NOP1e, containing the intron between the second and
third coding exons (exons 5 and 6) was reported in mice
(Pan et al., 1998b), rats (Chen et al., 1994; Xie et al.,
1999, 2000), and human brain (Xie et al., 1999). The
initial report in rats found an 84-bp insertion that could
be translated through to generate a full-length receptor.
The mouse version, however, had only 81 bp, and the
presence of a stop codon prevented translation of the last
three transmembrane domains (Pan et al., 1998a). A
more recent report in rats found a similar 81-bp insertion with a stop codon (Xie et al., 1999). It is not clear
which of the two rat variants predominates, but this is
an important issue since one has the potential of being a
functional variant whereas the other does not.
An additional three NOP1 variants have been described that contain mini-exons located between the first
and second coding exons (exons 2 and 5) (Fig. 2B) (Xie et
al., 1999). NOP1a contains a 34-bp insertion (exon 3)
between the first two coding exons. Due to a frameshift,
it gives a predicted stop codon in exon 5, yielding a
truncated protein lacking the seven transmembrane domains typically associated with G-protein-coupled receptors. NOP1c contains a different mini-exon insertion
of 139 bp (exon 4) and predicts a truncated protein due
to a frameshift and a predicted stop-codon. The other
variant, NOP1b, shows a different splice site within exon
4, the same mini-exon as NOP1c, and contains only the
3⬘ portion of the mini-exon (98 bp). Like the others, it
also gives a predicted truncated protein. The significance of these truncated proteins is still not fully understood. Clearly, they do not function like traditional Gprotein-coupled receptors. Yet, this does not necessarily
imply that they have no functional significance. It is
interesting that similar truncated variants have been
reported for all the other opioid receptor genes.
C. Molecular Modifications of NOP1
Although NOP1 itself has little affinity for traditional
opioids, it can be converted into an opioid-like receptor
either by simple mutations or by generating chimeras.
In the rat NOP1, a series of mutations within the transmembrane regions that had little effect upon the affinity
of OFQ/N itself markedly transformed the affinity of the
receptor up to 50-fold for dynorphin A and several of its
analogs, although the mutants still did not show appreciable affinity for ␤-endorphin (Meng et al., 1996). These
mutations were dispersed throughout the protein, including an A213K mutation in TM5, a triple mutation
VQV276 –278IHI in TM6, and a T302I mutation near
the top of TM7. When the T302I and VQV276 –278IHI
mutations were combined, the affinity of dynorphin A
increased even further, with a Ki under 1 nM.
Chimeras also illustrate the close relationship between the NOP1 receptor and the traditional opioid receptors. Chimeras combining the NOP1 and KOP1 (originally termed KOR-1) receptors were able to maintain
high affinity for OFQ/N and dynorphin A (Lapalu et al.,
1998; Mollereau et al., 1999). The first coding exon of the
NOP1 receptor encodes the first transmembrane domain, just as in the traditional opioid receptors. Exchanging the first coding exon of the NOP1 receptor with
the first exon of the ␮ opioid receptor MOP1 (originally
termed MOR-1) or the ␦ opioid receptor DOP1 (originally
termed DOR-1) did not appreciably affect the affinity of
the receptor for OFQ/N, but it did enhance the affinity of
the ␬3 ligand naloxone benzoylhydrazone (NalBzoH) approximately 5-fold and diminished the affinity of the
truncated OFQ/N derivative OFQ/N(1–11) (Pan et al.,
1996c). More interesting, however, the exchange with
the first exon of DOP1 converted NalBzoH from an agonist into an antagonist without changing its affinity.
Opiates such as morphine still did not show appreciable
affinity for any of the chimeras.
III. Receptor Binding
Structurally, the NOP1 receptor has the anticipated
seven transmembrane domains expected from members
of the G-protein-coupled class of receptors. NOP1 binding is sensitive to sodium and divalent cations (Ardati et
al., 1997), as first described with opioid receptors (Pert
and Snyder, 1973; Pasternak et al., 1975a,b; Wilson et
al., 1975), and OFQ/N and its analogs modulate
guanosine 5⬘-3-O-(thio)triphosphate binding in a pertussis toxin-sensitive manner (Reinscheid et al., 1995,
1996; Knoflach et al., 1996; Shimohira et al., 1997; Sim
and Childers, 1997; Meis and Pape, 1998; Narita et al.,
1999). The first description of OFQ/N binding to NOP1
utilized an iodinated analog, 125I-[Tyr14]OFQ/N (Reinscheid et al., 1995). Although many laboratories have
used this ligand, others have used 3H-OFQ/N (Dooley
and Houghten, 1996), which yields results virtually
identical to those of the iodinated ligand (Ardati et al.,
1997). More recently, a novel radioligand for the NOP1
receptor, 3H-ac-RYYRWK-NH2, was reported (Thomsen
et al., 2000).
Most groups found a high affinity of OFQ/N for the
transfected NOP1 receptor, typically around 50 pM, although there is a moderately wide range of values that
MOGIL AND PASTERNAK
likely reflect differences in assay techniques, buffers,
and cell lines (Dooley and Houghten, 2000) and ligand
stability (Quigley et al., 2000). Yet, there is good agreement regarding the specificity of the labeling, which
clearly distinguished the NOP1 receptor from traditional
opioid receptors. Of a wide variety of opiates, only NalBzoH showed a significant affinity for the site (Ki ⫽ 310
nM; Table 2), and even this was far less than its potency
at opioid sites (Ki ⬍ 10 nM). Morphine and other alkaloids have Ki values well above 1000 nM.
The initial characterization of NOP1 binding studies
examined the interactions of OFQ/N and its analogs
with the cloned receptor. Subsequent studies on brain
tissue have yielded more diverse results. Although several laboratories found KD values in brain similar to
those in transfected cell lines (Albrecht et al., 1998;
Nicholson et al., 1998; Thomsen et al., 2000), a number
of other groups report far lower affinities (Dooley and
Houghten, 1996, 2000; Wu et al., 1997; Mathis et al.,
1998, 1999). Wide variations of binding levels also were
reported. For example, reports of binding in rat cortex
range from 22 fmol/mg of protein (Thomsen et al., 2000)
to 291 fmol/mg of protein (Albrecht et al., 1998). The
reasons underlying these differences are not clear. However, there are a number of variables that may play a
role that include the choice of radioligand and binding
conditions (Dooley and Houghten, 2000).
IV. NOP1 Receptor Heterogeneity
A major question regarding the NOP1 receptor involves binding site heterogeneity. Evidence raising the
possibility of multiple OFQ/N receptors comes from both
pharmacological and binding sources. As noted earlier,
the Oprl1 gene that encodes the NOP1 receptor undergoes alternative splicing, but none of the additional variants identified to date encode a full-length receptor.
Although the combined evidence for multiple classes of
OFQ/N binding sites is suggestive, none of the studies
provides conclusive evidence for NOP1 receptor heterogeneity. Yet, it is worthwhile reviewing the evidence.
The behavioral pharmacology of OFQ/N is reviewed in
detail in subsequent sections. However, several issues
played a major role in exploring the possibility of mulTABLE 2
Competition of [Tyr14]OFQ/N binding to the cloned NOP1 receptor in
CHO cells
Ligand
Ki values
OFQ/N
[Tyr14]OFQ/N
OFQ/N(1–7)
OFQ/N(1–11)
NalBzoH
Morphine
U50,488H
DPDPE
Diprenorphine
0.088 ⫾ 0.007
0.071 ⫾ 0.019
⬎1000
55 ⫾ 22
310 ⫾ 75
⬎1000
⬎1000
⬎1000
⬎1000
nM
Data taken from Pan et al., 1996c.
385
tiple OFQ/N receptors. As discussed below, OFQ/N reportedly has both hyperalgesic and analgesic activities.
The hyperalgesic activity was insensitive to opioid antagonists, as expected based upon their poor affinity for
the OFQ/N binding sites. However, several laboratories
have observed that opioid antagonists reverse the analgesic responses of OFQ/N (Rossi et al., 1996b, 1997,
1998b; Jhamandas et al., 1998; Kolesnikov and Pasternak, 1999). Many assumed that OFQ/N activated a neural circuit with a downstream opioid link, which could be
blocked by the antagonists, and this is still a consideration. However, the opioid antagonist Win44,441 blocks
the inhibition of cAMP accumulation produced by
OFQ/N in mouse brain (Mathis et al., 1997). Since this
assay directly measures the effects of the receptor coupled to cyclase in membrane fragments, it is difficult to
envision how the antagonist could act other than directly blocking the receptor activated by OFQ/N. Thus,
this OFQ/N action in brain membranes argues for an
opioid antagonist-sensitive OFQ/N receptor.
Antisense mapping studies also differentiated between OFQ/N analgesia and hyperalgesia. Whereas
probes targeting the second and third coding exons of
the NOP1 receptor down-regulated OFQ/N and NalBzoH
analgesia (King et al., 1997; Rossi et al., 1997), a probe
targeting the first coding exon was ineffective. Yet, the
same antisense probe based upon the first coding exon
blocked the hyperalgesia and anti-opioid activity of
OFQ/N, whereas the antisense probes targeting exons 2
and/or 3 that were active against OFQ/N analgesia had
no effect against hyperalgesia (King et al., 1997; Rossi et
al., 1997, 1998). Although these pharmacological assays
are suggestive, behavioral approaches have many potential subtleties and alternative explanations. Thus, they
do not provide conclusive evidence for multiple OFQ/N
receptors.
Binding studies also suggest binding site heterogeneity. Early studies with 125I-[Tyr14]OFQ/N in mouse
brain revealed curvilinear Scatchard plots, suggestive of
sites of differing affinity (Mathis et al., 1997). However,
this does not necessarily imply different receptors. In
NOP1-transfected HEK293 cells with high levels of expression, 3H-OFQ/N binding also yielded biphasic Scatchard plots, presumably reflecting two conformations of
the receptor (Ardati et al., 1997). Alternatively, curvilinear Scatchard plots can result from radioligand degradation. Saturation studies alone cannot distinguish
among these possibilities and must be interpreted in
conjunction with other types of studies. However, other
approaches also implied NOP1 receptor binding heterogeneity.
OFQ/N(1–11) is a truncated peptide derived from
OFQ/N. In vivo, it is functionally active, eliciting analgesia (Rossi et al., 1997) and inhibiting cAMP accumulation in brain membranes (Mathis et al., 1997). These
actions were not anticipated based upon its very poor
affinity for the cloned NOP1 receptor in binding assays.
386
To more accurately assess the possibility of a novel OFQ/
N(1–11) binding site, tyrosine-containing analogs were
generated that could be iodinated and used to examine
binding sites directly (Mathis et al., 1998), as done earlier with OFQ/N itself (Reinscheid et al., 1995). Of the
analogs, [Tyr10]OFQ/N(1–11) proved most valuable.
Like OFQ/N(1–11), [Tyr10]OFQ/N(1–11) is analgesic
when given supraspinally in mice and it competes 125I[Tyr14]OFQ/N binding in mouse brain more potently (Ki
⫽ 79 nM) than OFQ/N(1–11) itself (Ki ⫽ 262 nM). Iodinating the analog to iodo[Tyr10]OFQ/N(1–11) further
enhanced its potency (Ki ⫽ 39 nM).
In mouse brain, 125I-[Tyr10]OFQ/N(1–11) labeling
strongly suggested a novel binding site distinct from the
binding of 125I-[Tyr14]OFQ/N. Binding parameters of
125
I-[Tyr10]OFQ/N(1–11) revealed an affinity (KD) of
0.24 nM, which is over 100-fold lower than its Ki against
125
I-[Tyr14]OFQ/N binding in mouse brain and more
than 10-fold lower than its KD determined in CHO cells
transfected with the NOP1 receptor. Furthermore, in
brain it displayed a Bmax of only 43 fmol/mg of protein,
which is far fewer sites than observed in companion
assays with 125I-[Tyr14]OFQ/N (Table 3) or from the
literature (Dooley and Houghten, 1996; Albrecht et al.,
1998; Nicholson et al., 1998). It also is interesting that
the capacity of the 125I-[Tyr10]OFQ/N(1–11) site is similar to the higher affinity (KD ⫽ 4 pM) site observed in
mouse brain for 125I-[Tyr14]OFQ/N. A possible association of the two is further suggested by saturation studies
with 125I-[Tyr14]OFQ/N in which the inclusion of OFQ/
N(1–11) appeared to selectively reduce the higher affinity binding component of 125I-[Tyr14]OFQ/N.
The difference in selectivity between OFQ/N(1–11)
and the standard OFQ/N radioligands was quite revealing (Mathis et al., 1999). OFQ/N and its analogs labeled
the 125I-[Tyr10]OFQ/N(1–11) site with very high affinity,
confirming its classification as an OFQ/N site. The affinity of OFQ/N(1–11) increases about 30-fold, with its
Ki dropping to only 8.7 nM. It is interesting, however,
that the affinity of OFQ/N(1–7) is unchanged and remains quite poor.
As previously noted, 125I-[Tyr14]OFQ/N binding is insensitive to opioids. In contrast, 125I-[Tyr10]OFQ/N(1–
11) binding is competed by a wide variety of opioid
ligands. Although the affinities of most of the opioids
TABLE 3
Saturation analysis of 125I-[Tyr14]OFQ/N and 125I-[Tyr11]OFQ/N(1–11)
binding in transfected CHO cells and mouse brain
Radioligand
125
I-[Tyr10]OFQ/N(1–11)
I-[Tyr14]OFQ/N
Site 1
Site 2
125
NOP1/CHO
Cells
Mouse Brain
KD (pM)
KD (pM)
Bmax (fmol/
mg protein)
2700 ⫾ 700
235 ⫾ 7.8
43.2 ⫾ 4.6
37 ⫾ 2
Not applicable
3.8 ⫾ 3.3
896 ⫾ 636
32 ⫾ 18
233 ⫾ 154
Data adapted from Mathis et al., 1999.
examined remain lower than against traditional opioid
receptors, a number of compounds showed high affinity
for this site (Table 4). Among the opiates, NalBzoH was
the most impressive. In brain membranes, its affinity
against 125I-[Tyr10]OFQ/N(1–11) binding (Ki ⫽ 3.9 nM)
is similar to that seen with traditional opioid binding
sites and almost 100-fold greater than 125I-[Tyr14]OFQ/
N binding. The affinity of fentanyl is increased over
100-fold against the OFQ/N(1–11) site. Some opioid peptides also show high affinity for the 125I-[Tyr10]OFQ/
N(1–11) site, particularly dynorphin A and ␣-neoendorphin. Indeed, dynorphin A labels the 125I-[Tyr10]OFQ/
N(1–11) site as potently as ␮ and ␦ opioid receptors.
Together, along with the dramatic anatomical differences between 125I-[Tyr14]OFQ/N and 125I-[Tyr10]OFQ/
N(1–11) binding (see below), these studies suggest the
possibility of OFQ/N receptor heterogeneity. If multiple
OFQ/N sites exist, they may correspond to splice variants of NOP1 receptor. Alternatively, they might correspond to post-translational modifications of the receptors, result from modulation of the receptor by
additional proteins, or be expressed by a totally different
gene. However, without more definitive biochemical evidence, OFQ/N binding site heterogeneity remains tentative.
V. Orphanin FQ/Nociceptin
A. Structure of Orphanin FQ/Nociceptin
The unusual properties of the “orphan opioid receptor”
soon led to the identification of an endogenous peptide,
termed orphanin FQ (Reinscheid et al., 1995) or nociceptin (Meunier et al., 1995) (OFQ/N), that labeled the
cloned receptor with very high affinity (Fig. 1). OFQ/N is
a heptadecapeptide with some interesting structural homologies to the classical opioid peptide dynorphin A
(Fig. 1). Both peptides are comprised of 17 amino acids
bounded by pairs of basic amino acids important in their
production from their precursors. Furthermore, both
have internal pairs of basic amino acids, raising the
possibility of further processing. The opioid peptides
share a YGGF motif, where the fifth amino acid is either
leucine or methionine. The amino terminus of OFQ/N is
a phenylalanine instead of a tyrosine, followed by GGF.
Finally, both peptides contain the same last two amino
acids at the carboxyl terminus. Despite these similarities, the peptides are functionally quite distinct. OFQ/N
has no appreciable affinity for any of the opioid receptors. Alanine scanning reveals the importance of the
amino acids in positions 1, 2, 4, and 8 (Dooley and
Houghten, 1996). Of these, the phenylalanine in position
1 is particularly important in establishing the selectivity
of binding since replacing it with a tyrosine yields analogs with far greater affinity at traditional opioid receptors, although the [Tyr1] analog still can induce naloxone-insensitive actions presumably mediated through
NOP1 receptors (Champion and Kadowitz, 1997a,b). The
387
MOGIL AND PASTERNAK
Competition of
125
TABLE 4
I-[Tyr10]OFQ/N(1–11) binding in mouse brain membranes
125
I-[Tyr14]OFQ
125
I-[Tyr10]OFQ/N(1–11)
Ratio
Drug
Ki
Hill Slope
nM
OFQ/N and related
peptides
OFQ/N
[Tyr14]OFQ/N
Iodo[Tyr14]OFQ/N
OFQ/N (1–11)
[Tyr10]OFQ/N (1–11)
Iodo[Tyr10]OFQ/N (1–11)
OFQ/N (1–7)
Opioid peptides
Dynorphin A (1–17)
Dynorphin B (1–13)
␣-Neoendorphin
[Met5]Enkephalin
[Leu5]Enkephalin
DAMGO
DPDPE
Opiates
Morphine
Naltrindole
Nor-binaltorphimine
Naltrexone
Naloxone
NalBzoH
Fentanyl
(⫺)Cyclazocine
(⫹)Cyclazocine
Levallorphan
Diprenorphine
(⫺)Pentazocine
(⫹)Pentazocine
U50,488H
Hill Slope
nM
0.17 ⫾ 0.06
0.65 ⫾ 0.36
0.12 ⫾ 0.03
262 ⫾ 80
79.8 ⫾ 10.1
38.9 ⫾ 5.9
360 ⫾ 110
0.60
0.43
0.93
0.67
0.91
0.93
0.70
72.8 ⫾ 22
247 ⫾ 10
192 ⫾ 14
⬎1000
⬎1000
⬎1000
⬎1000
0.98
1.1
1.3
⬎1000
⬎5000
⬎7500
⬎1000
⬎1000
358 ⫾ 57
⬎5000
⬎7500
⬎1000
⬎5000
⬎4000
⬎1000
⬎1000
⬎1000
Ki
0.79
0.007 ⫾ 0.002
0.007 ⫾ 0.0003
0.006 ⫾ 0.002
8.7 ⫾ 1.9
1.6 ⫾ 0.4
0.58 ⫾ 0.16
440 ⫾ 134
6.0 ⫾ 0.7
33.9 ⫾ 8.3
6.5 ⫾ 1.5
⬎1000
⬎1000
⬎1000
⬎1000
⬎1000
236 ⫾ 74
898 ⫾ 217
⬎1000
⬎1000
3.9 ⫾ 0.2
39.6 ⫾ 5.0
157 ⫾ 11.5
⬎1000
266 ⫾ 12
312 ⫾ 76
⬎1000
⬎1000
⬎1000
0.73
1.1
0.78
0.73
1.2
0.88
0.83
24
93
20
30
50
67
0.8
0.88
0.74
0.89
12
7
30
0.76
0.58
⬎21
⬎8.4
0.99
0.82
0.84
92
⬎126
⬎48
0.52
0.55
⬎19
⬎13
Data adapted from Mathis et al. (1997, 1999).
basic structure can be modified and even truncated at its
carboxyl terminus without major loss of activity, but the
initial FGGF motif is required for activity (Guerrini et
al., 1997).
B. Orphanin FQ/Nociceptin Analogs and Antagonists
Full descriptions of all the structure-activity relationships of OFQ/N is beyond the scope of this review. However, several analogs are important. The first analog,
[Tyr14]OFQ/N, was developed to enable the detection of
receptor binding and has been particularly important
(Reinscheid et al., 1995). Replacing the leucine at position 14 yielded a peptide that could be iodinated and still
maintain affinity for the receptor similar to the parent
compound (Reinscheid et al., 1995). This analog has
proven extremely valuable in the characterization of the
receptor in both transfected cell lines and in the brain.
OFQ/N has two pairs of basic amino acids within its
structure, raising the possibility of further processing to
yield OFQ/N(1–11) and OFQ/N(1–7). Although there are
studies showing the activity of these peptides and suggesting that their pharmacology may differ from that of
OFQ/N itself (Rossi et al., 1997), as described below, the
physiological significance of these truncated peptides
has not been fully established. The possibility that the
truncated peptides also might be relevant led to the
development of a tyrosine-containing analog of OFQ/
N(1–11) suitable for iodination (Mathis et al., 1998).
Analogs were synthesized with tyrosine at positions 1,
10, or 11. The placement of tyrosine at position 1 lowered
its affinity against NOP1 binding in transfected cells,
but enhanced its potency against the traditional opioid
receptors. [Tyr11]OFQ(1–11) and its iodinated version,
iodo[Tyr11]OFQ/N(1–11), on the other hand, were devoid
of activity against traditional opioid receptors and more
potent against NOP1 binding in transfected cells than
OFQ/N(1–11) itself. Both [Tyr11]OFQ(1–11) and
iodo[Tyr11]OFQ/N(1–11) were pharmacologically active,
eliciting analgesia in mice. As discussed earlier, these
agents have proven valuable in binding studies.
The evaluation of the pharmacology of OFQ/N was hindered for a number of years by the lack of an effective antagonist. The first proposed antagonist, [Phe1␺(CH2-NH)Gly2]nociceptin(1–13)-NH2, was subsequently found to be a partial
agonist, with many groups observing OFQ/N-like actions in a
variety of models. A recently described small molecule antagonist, J-113397 (1-[3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2one) (Kawamoto et al., 1999; Ozaki et al., 2000a,b), has
proven valuable in a number of models (see Section VIII.G.2.).
388
C. Orphanin FQ/Nociceptin Precursors and Their
Processing
The OFQ/N sequence contains pairs of basic amino
acids that might imply additional processing of the peptide to OFQ/N(1–11) and/or OFQ/N(1–7). Both of these
truncated peptides are functionally active when administered in vivo (Rossi et al., 1997), producing analgesia
that is reversed by opioid antagonists. Neither peptide
shows appreciable affinity for any of the traditional opioid receptors, but OFQ/N(1–11) does label cloned NOP1
receptors moderately well (Ki ⫽ 55 nM), although its
affinity still is far lower than OFQ/N itself. OFQ/N(1–7)
does not compete with binding to the NOP1 receptor at
doses as high as 1 ␮M. The true significance of these
peptides remains to be demonstrated.
Like most neuropeptides, OFQ/N is generated from a
larger precursor peptide, prepro-OFQ/N (ppOFQ/N) that
has been cloned from mouse, rat, and human (Fig. 3)
(Meunier et al., 1995; Pan et al., 1996a; Reinscheid et al.,
2000) and that has been localized in man to chromosome
8 (8p21) (Mollereau et al., 1996). Overall, there is high
interspecies homology, with 80% identity among the
three organisms. Within the precursor, there are several
additional peptides suggested by the presence of pairs of
basic amino acids. Nocistatin has been examined most
extensively (see Section IX.A.). Nocistatin possesses analgesic actions and presumably acts through a distinct
receptor since it has no appreciable affinity for any of the
traditional opioid receptors or NOP1. It is interesting
that the nocistatin sequence shows the most variability
of the putative peptides within ppOFQ/N among species.
The mouse version is the longest, containing 41 amino
acids, whereas the rat peptide has 35 and the human
form only 30. The mouse sequence has an interesting
DAEPGA motif that is repeated three times. The rat
form has a similar double repeat, but the human form
does not. The differences between the species rests primarily over the length of this repeat, with the human
form lacking 10 of the amino acids of the mouse version
at this location.
Another peptide was predicted from the sequence of
ppOFQ/N based upon the presence of pairs of basic
amino acids suggesting sites of peptide processing. Orphanin FQ2 is a heptadecapeptide, like OFQ/N and
dynorphin A, with a phenylalanine (F) and glutamine
(Q) at the first and last position, leading to its name,
OFQ2 (also called NocII; and hereinafter called OFQ2/
NocII). The placement of OFQ2/NocII within ppOFQ/N
is interesting in that OFQ2/NocII is immediately downstream from OFQ, much like dynorphin B is immediately downstream of dynorphin A in preprodynorphin.
When administered centrally, OFQ2/NocII is pharmacologically active, raising the possibility that it is physiologically relevant (Rossi et al., 1998a; Florin et al., 1999)
(see Section IX.B.). A longer peptide containing the
OFQ2/NocII sequence at its amino terminus, ppOFQ/
N(180 –187), has been described and it also is functionally active in mice (Mathis et al., 2001). It is still an open
question as to whether ppOFQ/N(180 –187) is active itself or whether it is further processed to OFQ2/NocII.
VI. Anatomy of Orphanin FQ/Nociceptin and Its
Receptor
The regional distribution of OFQ/N and the NOP1
receptor have been well described (Bunzow et al., 1994;
Fukuda et al., 1994; Mollereau et al., 1994; Wick et al.,
1994; Lachowicz et al., 1995; Nothacker et al., 1996;
Riedl et al., 1996; Houtani et al., 2000; Neal et al.,
1999a,b; Letchworth et al., 2000; O’Donnell et al., 2001).
These series of publications provide detailed descriptions of the distribution of the NOP1 receptor mRNA and
binding in the brain which are beyond the scope of this
review. Overall, they report a good correlation between
receptor binding distributions and those seen with in
situ hybridization. Regions with NOP1 receptor binding
typically express NOP1 mRNA as well, although the
levels of mRNA and binding do not always match very
closely. Regions with high levels of NOP1 binding/mRNA
include the cortex, anterior olfactory nucleus, lateral
septum, hypothalamus, hippocampus, amygdala, cen-
FIG. 3. Schematic of the prepro-OFQ/N gene.
MOGIL AND PASTERNAK
389
FIG. 4. Autoradiographic studies of 125I-[Tyr14]OFQ and 125I-[Tyr10]OFQ(1–11) in rat brain. Sections of rat brain were incubated with either
I-[Tyr14]OFQ or 125I-[Tyr10]OFQ(1–11) and opposed to film. The exposure time was longer for 125I-[Tyr10]OFQ(1–11) than for 125I-[Tyr14]OFQ due
to the lower binding levels. Reprinted from Letchworth et al., 2000.
125
tral gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, and spinal
cord. The distribution patterns have suggested the involvement of the NOP1 receptor system in “motor and
balance control, reinforcement and reward, nociception,
the stress response, sexual behavior, aggression and
autonomic control of physiological processes” (Neal et
al., 1999a).
The distribution of OFQ/N also has been well described in the literature (Dickenson, 1996; Riedl et al.,
1996; Kummer and Fischer, 1997; Mitsuma et al., 1998;
Neal et al., 1999b; Houtani et al., 2000; O’Donnell et al.,
2001). In brief, the localization of OFQ/N corresponds
reasonably well with the NOP1 receptor. As with the
receptor, OFQ/N immunoreactivity and mRNA levels
detected using in situ hybridization are closely correlated. OFQ/N is found in lateral septum, hypothalamus,
ventral forebrain, claustrum, mammillary bodies, amygdala, hippocampus, thalamus, medial habenula, ventral
tegmentum, substantia nigra, central gray, interpeduncular nucleus, locus coeruleus, raphe complex, solitary
nucleus, nucleus ambiguous, caudal spinal trigeminal
nucleus, and reticular formation, as well the ventral and
dorsal horns of the spinal cord (Neal et al., 1999b).
The distribution of 125I-[Tyr10]OFQ/N(1–11) in the
brain also is distinct autoradiographically (Fig. 4)
(Letchworth et al., 2000). The distribution of 125I[Tyr14]OFQ/N binding was described earlier. 125I[Tyr10]OFQ/N(1–11) binding also shows intense labeling
of the cortex, but far lower levels of labeling in deeper
structures. Compared with 125I-[Tyr14]OFQ/N, 125I[Tyr10]OFQ/N(1–11) labeling is far less intense in the
olfactory tubercle, nucleus accumbens, striatum, lateral
and medial septum, hypothalamus, as well as a number
of brain stem structures such as the periaqueductal
gray, medial raphe, and locus coeruleus.
VII. Range of Effects of Orphanin FQ/Nociceptin
Befitting its particularly wide distribution in the nervous system (see above), there are a myriad of proposed
functional roles for OFQ/N. Receiving by far the most
attention is the involvement of this peptide in the mediation and modulation of pain in the supraspinal, spinal, and peripheral compartments of the nervous system. Related proposed functions for OFQ/N include roles
in opiate tolerance, dependence/withdrawal, and adaptive responses to anxiety and stress. However, studies
390
TABLE 5
Systems-level biological phenomena in which OFQ/N has been implicated
Phenomenon
Anxiety
Cardiovascular
Circadian rhythms
Dependence
Feeding
Hearing
Gastrointestinal function
Immunity
Inflammation
Learning (spatial)
Locomotion
Long-term potentiation
Memory (passive avoidance)
Pain
Renal function
Reward (cocaine)
Reward (ethanol)
Reward (morphine)
Respiration
Sexuality
Thermoregulation
Tolerance
Vestibular functioning
Effect of OFQ/N Administration
Anxiolytic
Anti-arrhythmic
Bradycardia
Hypotension
Vasodilatation (cerebral)
Vasodilatation (peripheral)
Blocks light-induced phase shift
Induces withdrawal symptoms
Hyperphagia
Unknownb
Stimulates motility (in rat)
Inhibits motility (in mouse)
Inhibits surgical immunosuppression
Increases vascular permeability
Impairment
Hypo- or hyperlocomotion
Impairment
Impairment
Controversial
Diuresis, antinatriuresis
Inhibits micturition reflex
Induces sensitization
Blocks intake, place preference
Blocks place preference
Inhibits bronchoconstriction
Facilitates lordosis
Facilitates erection
Hypothermia
Unknownb
Decreases vestibulo-ocular reflex gain
Referencea
See Section X.B.
Maslov et al., 1999
Salis et al., 2000
Salis et al., 2000
Armstead, 1999
Salis et al., 2000
Allen et al., 1999
See Section X.C.
See Section X.E.
Nishi et al., 1997
Osinski and Brown, 2000
Osinski and Brown, 2000
Du et al., 1998
Kimura et al., 2000
See Section X.D.
See Section X.A.
See Section X.D.
See Section X.D.
See Section VIII.
Kapusta, 2000
Lecci et al., 2000a
See Section X.A.
See Section X.A.
See Section X.A.
Peiser et al., 2000
Sinchak et al., 1997
Champion et al., 1997
Yakimova and Pierau, 1999
See Section X.C.
Sulaiman et al., 1999
a
Not exhaustive; the most recent review of the subject is cited where available.
b
The involvement of OFQ/N in these phenomena has been suggested via experimental approaches other than the direct injection of the peptide (e.g., antibody/antisense/
antagonist administration, knockout mouse phenotype).
based on direct injection of the peptide, measurement of
peptide levels, administration of antagonist/antisense
compounds and/or the evaluation of the phenotype of
transgenic “knockout” mutants have implicated OFQ/N
in the mediation of biological phenomena ranging from
learning and memory to hearing to water balance to
reproductive physiology. A list of OFQ/N-associated systems-level phenomena is presented in Table 5. Some of
the more well studied and noteworthy phenomena will
be discussed presently, starting with pain processing.
VIII. Effects of Orphanin FQ/Nociceptin on Pain
A. Effects of Supraspinally Administered Orphanin
FQ/Nociceptin
The first in vivo action of OFQ/N reported by both its
discoverers was a reduction in latency to respond to
noxious thermal stimuli on the tail-flick (Reinscheid et
al., 1995) and hot-plate tests (Meunier et al., 1995) after
supraspinal (intracerebroventricular) injection in the
mouse. Both groups interpreted these data as reflective
of a hyperalgesic action; i.e., a decrease in nociceptive
threshold (increase in nociceptive sensitivity) produced
by the peptide. This was very much a surprise, since
classical opioids, with the possible exception of dynorphin (see Caudle and Mannes, 2000), produce analgesic
and/or antihyperalgesic effects (see Pasternak, 1993).
The apparent hyperalgesia produced by supraspinal
OFQ/N inspired Meunier and colleagues (1995) to dub
the peptide nociceptin.
Exogenous administration of an endogenous compound is not an ideal method for gleaning its true physiological role. When injected intracerebroventricularly,
OFQ/N will be widely dispersed throughout the ventricular system, possibly affecting populations of ORL1 receptors that would not normally be activated by endogenously released peptide. Tissue levels are dependent
upon diffusion of the agent from the cerebrospinal fluid
into the brain, which results in a decreasing gradient of
drug concentrations in deeper structures. The drug even
can diffuse to spinal sites, particularly with high injection volumes. This makes it difficult to judge the concentration of peptide in relevant brain loci and thereby
assess whether its concentration is appropriate or
grossly supraphysiological. Finally, this approach entirely ignores contextual elements accompanying
OFQ/N release under usual circumstances. Nonetheless,
in the absence of an ORL1 antagonist and with the vast
majority of the studies reviewed herein conducted before
any such antagonist was available, direct injection of
OFQ/N was one of only a handful of feasible experimental approaches.
In contrast to the conclusions from the initial descriptions of OFQ/N, we now recognize that there is no widely
accepted “role” of OFQ/N in supraspinal pain-modulatory circuits. In fact, even the effects of supraspinal
MOGIL AND PASTERNAK
projection of OFQ/N on nociceptive sensitivity remain
highly contentious. As detailed in Table 6, reports in the
literature have suggested six different “effects” of supraspinal OFQ/N on nociception: 1) hyperalgesia, 2) analgesia, 3) hyperalgesia followed by analgesia, 4) neither
hyperalgesia nor analgesia, 5) anti-analgesia but not
hyperalgesia, and 6) anti-analgesia plus hyperalgesia.
The only uncontested observation is the anti-analgesic
activity of OFQ/N, first documented in 1996 (Mogil et al.,
1996a)
OFQ/N blocks analgesia from a wide variety of exogenous and endogenous opioid compounds. Since
OFQ/N has negligible affinity for any of the traditional opioid receptors, it must act through neural
circuits as a “functional antagonist”, rather than
through direct molecular interactions with opioid receptors. Given intracerebroventricularly, OFQ/N can
reverse and/or prevent analgesia from drugs acting at
supraspinal ␮-opioid receptors, including morphine
(Grisel et al., 1996; Mogil et al., 1996a; Tian et al.,
1997b; Zhu et al., 1997; Calo’ et al., 1998; King et al.,
1998; Lutfy et al., 1999; Citterio et al., 2000), DAMGO
(Mogil et al., 1996b), fentanyl (Zhu et al., 1998), acetorphan (Suaudeau et al., 1998), endomorphin-1
(Wang et al., 1999a,c), and morphine-6␤-glucuronide
(King et al., 1998). It has similar effects against supraspinal ␦-opioid agonists, like DPDPE (Mogil et al.,
1996b; King et al., 1998) and DSLET (Zhu et al., 1998;
Wang et al., 1999a), ␬1-opioid agonists like U50,488
(Mogil et al., 1996b; Zhu et al., 1998; Wang et al.,
1999a), and dynorphin A (Citterio et al., 2000), and
the ␬3-opioid agonist, naloxone benzoylhydrazone
(King et al., 1998).
Direct injections of OFQ/N into specific brain loci also
induce anti-analgesic actions. OFQ/N placed into the
periaqueductal gray (PAG) blocks morphine analgesia
(Morgan et al., 1997) and its administration into the
rostral ventromedial medulla (RVM) reverses DAMGO
analgesia (Heinricher et al., 1997; Pan et al., 2000) (see
Section VIII.H.). Importantly, OFQ/N also blocks analgesia from endogenous opioid-mediated manipulations,
including electroacupuncture (Zhu et al., 1996; Tian et
al., 1997a; Zhang et al., 1997) and mild stressors (Mogil
et al., 1996a; Suaudeau et al., 1998; Rizzi et al., 2001).
The latter phenomenon may be responsible for much of
the confusion surrounding the actions of OFQ/N (see
Section VII.D.3.).
The anti-opioid effect of OFQ/N against morphine analgesia is long-lasting, persisting for up to 4 to 6 h
(Candeletti and Ferri, 2000). Repeated OFQ/N dosing
induces tolerance, with a decreasing response over time
(Lutfy et al., 1999). Although it is tempting to only
assume a functional interaction between OFQ/N and
other members of the opioid gene family, the anti-analgesic actions of this peptide are by no means restricted to
opioid analgesia. OFQ/N equally efficaciously blocks analgesia from the ␣2-adrenergic receptor agonist,
391
clonidine (King et al., 1998), the GABAB receptor agonist, baclofen (Citterio et al., 2000), and naloxone-insensitive forms of swim stress (Rizzi et al., 2001).
This ability to block non-opioid analgesia sets OFQ/N
apart from other known functional anti-opioid peptides,
including adrenocorticotrophic hormone (ACTH), cholecystokinin (CCK), dynorphin, FMRFamide (and its analogs), ␣-melanocyte-stimulating hormone (␣-MSH),
MIF-1/Tyr-MIF-1, neurotensin- and tyrosine-releasing
hormone (Rothman, 1992), and ␴1 receptor systems (e.g.,
Chien and Pasternak, 1993). Anti-opioid systems are
thought to play important roles in a number of painrelevant phenomena, including the mediation of individual differences in analgesic sensitivity (Chien and Pasternak, 1993; Tang et al., 1997), the induction of
tolerance and dependence (Rothman, 1992) (see Section
X.D.), and in plastic changes underlying neuropathic
pain (Wiesenfeld-Hallin et al., 1997). Elucidation of the
precise actions of OFQ/N vis-a`-vis these other anti-opioid peptides will be a major research challenge for the
future.
These anti-analgesic actions of OFQ/N are the most
robust activities observed following supraspinal administration, having been seen by all groups examining this
question. The two contentious issues, regarding OFQ/N
actions, that remain involve direct analgesia and hyperalgesia. In one study, for example, higher OFQ/N doses
induced analgesia in mice, although this action is not
easily detected in all strains (Rossi et al., 1997). In this
study, an initial hyperalgesic response was followed by
analgesia. The analgesic response was reversed by opioid antagonists, but the hyperalgesic actions were not.
Indeed, the biphasic hyperalgesic/analgesic activity seen
with OFQ/N alone reverted to only a monophasic hyperalgesia in the presence of the opioid antagonist. Others,
of course, see neither hyperalgesia or analgesia. Factors
relevant to interpreting the conflicting data presented in
Table 6 are discussed below.
A final comment concerns not the effect of OFQ/N on
pain, but the effect of pain on OFQ/N. A recent study
by Rosen and colleagues (2000) examined OFQ/N-like
immunoreactivity in various nociception-related brain
areas 2 weeks after the induction of a neuropathic
state using Bennett and Xie’s (1988) surgical model or
a carrageenan inflammatory model. Both injuries increased OFQ/N levels in the cingulate cortex, and
carrageenan increased levels also in the hypothalamus and the dorsal horn of the spinal cord. OFQ/N
levels did not change in the PAG or RVM (in contrast
to levels of dynorphin B and met-enkephalin-ArgPhe), prompting the authors to conclude that OFQ/N
is involved in the modulation of ascending nociceptive
transmission pathways rather than descending nociceptive modulation pathways (but see Section VIII.H.).
OFQ/N has also been identified in human cerebrospinal
fluid but not at higher levels in women with ongoing labor
pain compared with those presenting for elective Caesar-
392
TABLE 6
Parameters and results of published studies investigating the effect on nociceptive sensitivity of supraspinally (i.c.v.)-injected OFQ/N in rodents
Effect
Species
Straina
Sex
Assayb
Controlc
Intensityd
Dosee
Reference
nmol, i.c.v.
Hyperalgesia
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
Mouse
NMRI
CD-1
ICR
129xB6
129xB6
SD
ICR
么
么
N.R.
么
么
么
么
TWrh
HP
TWrh
TWrh
TWrh
TWrh
TWrh
Veh
Veh
Veh
Veh
Veh
All
Veh ⫹ BL
7s
65 sf
10 s
12 s
11 s
5.5 s
9s
ⱖ1
0.055
ⱖ0.005
10
10
1
ⱖ0.1
Reinscheid et al., 1995
Meunier et al., 1995
Shimohigashi et al., 1996
Nishi et al., 1997
Noda et al., 1998
Candeletti et al., 2000
Ozaki et al., 2000a
Mouse
Rat
CD-1
SD
么
么
TWrh
TWrh
BL
BL
2.5 s
2.5 s
5.5
ⱖ5.5
Rossi et al., 1996b
Rossi et al., 1998b
Mouse
CD-1
么
TWrh
Veh ⫹ BL
8.5 s
ⱖ5.5
Rossi et al., 1997g
Mouse
Rat
Rat
ICR
SD
SD
么
么
么⫹乆
TWhw/rh
TWhw
HP
Veh ⫹ BL
Veh ⫹ BL
Veh ⫹ BL
N.R.
N.R.
8–12 s
0.1–10
0.055–10
30
Mouse
SW
么⫹乆
Mouse
Rat
Mouse
Mouse
SW
Wistar
CD-1
CD-1
么⫹乆
么
么
么
Rat
Rat
Rat
Rat
Mouse
SD
SD
Lewis
SD
CD-1
么
乆
么
么
么
AC
HP
TWhw
TWhw
TWrh
TWrh
AC
HP
F
TWrh
HP
TWrh
CFAHT
TWrh
TWrh
Veh
Veh
All
Veh
Veh
Veh
Veh
Veh
Veh
Veh
Veh
Veh
Veh
Veh
Veh
0.6%
28 s
6.5 s
3.5 s
5s
2.5 s
0.6%
7s
2%, 30 ␮l
12 s
10 s
N.R.
13 s/7sk
5.5 s
3s
2.5
2.5
10
ⱖ5
ⱖ0.0004
ⱖ0.055
ⱖ0.55
0.055–0.14j
0.055j
ⱖ0.005
50
4.4
ⱖ1
2
ⱖ0.55
Rat
Rat
Rat
Mouse
Rat
Rat
Mouse
SD
SD
SD
Swiss
SD
SD
CD-1
么
么
么
么
么
N.R.
么
FS
TWrh
F
TWhw
F
TWhw
TWrh
Veh ⫹ BL
???
Veh
All
Veh
Veh ⫹ BL
Veh ⫹ NI
175 ␮A
???
5%, 60 ␮l
5s
5%, 150 ␮l
7s
13 s
ⱖ0.055
ⱖ0.055l
ⱖ0.055l
ⱖ0.1
ⱖ0.055l
ⱖ0.055l
ⱖ0.01l
Analgesia
Hyperalgesia, then
analgesia
No Hyperalgesia or
analgesia
Anti-analgesia, no
hyperalgesia
Hyperalgesia and
anti-analgesia
⫹ NI
⫹ NI
⫹ BL
⫹ BL
⫹ BL
⫹ BL
⫹ BL
Vanderah et al., 1998h
Lutfy and Maidment, 2000i
Mogil et al., 1996a
Grisel et al., 1996; Mogil et al., 1996b, 1998
Tian et al., 1997a,b
King et al., 1998
Suaudeau et al., 1998
Lutfy et al., 1999
Zhao et al., 1999
Bertorelli et al., 1999
Candeletti and Ferri, 2000
Rady et al., 2001
Zhu et al., 1996
Zhang et al., 1997; Zhu et al., 1998
Zhu et al., 1997
Calo’ et al., 1998
Wang et al., 1999a
Wang et al., 1999c
Citterio et al., 2000
N.R., not reported; ???, unknown.
Data adapted and updated from a similar table in Grisel and Mogil (2000).
a
Strain abbreviations: 129xB6, wild-type mouse on a mixed 129xC57BL/6 hybrid background; CD-1, Hsd:ICR; ICR, Institute for Cancer Research (Swiss-derived); NMRI,
a Swiss-derived European strain; SD, Sprague-Dawley; SW, Swiss-Webster.
b
Assay abbreviations: AC, abdominal constriction (writhing) test; CFAHT, complete Freund’s adjuvant-induced arthritis, with hyperalgesia measured on Hargreaves et
al. (1988) test of paw withdrawal from radiant heat; HP, hot-plate test; FS, electric footshock test; F, formalin test; TWhw, hot water tail-withdrawal test; TWrh, radiant
heat tail-withdrawal (tail-flick) test.
c
Control group abbreviations: All, all of the following were used; BL, comparison with baseline latencies; Veh, comparison with vehicle group; NI, comparison with
non-injected group. The use of a Veh control only may confound hyperalgesia with blockade of test-related stress-induced analgesia (i.e., anti-analgesia) (Mogil et al., 1996a).
Note that within-subject baseline values cannot be obtained in the AC and F tests.
d
For thermal assays, baseline latency or latency of control group is provided. For other assays, actual intensity parameters are listed. Test-related stress-induced
analgesia is more likely to be seen when mildly noxious stimuli are used (e.g., ⬎4 s in the TW test or ⬎15 s in the HP test).
e
Effective OFQ/N dose or dose range. Many of these studies investigated a wider overall dose range than that shown.
f
Latency to escape jumping. OFQ/N was also noted to reduce rearing latencies on the HP test in this study, but rearing is not a generally accepted nociceptive endpoint
in this test.
g
Significant hyperalgesia was observed 15 min post-injection. This effect wore off and was replaced by analgesia, which was significant at 60 min post-injection.
h
This is the overall conclusion of the authors of this study, although individual examples of possible hyperalgesia and analgesia were noted.
i
Hyperalgesia was revealed in this experiment at OFQ/N doses ⱖ15 nmol after pharmacological blockade of ␮ receptors (see Section VIII.D.6.).
j
Biphasic dose-response relationship observed.
k
Baseline latency of the contralateral (normal) and arthritic paw, respectively.
l
Dose at which anti-analgesic effects were noted. Hyperalgesia was only seen at higher doses.
ean section (Brooks et al., 1998). Thus, any clinical relevance of supraspinal OFQ/N remains to be demonstrated.
B. Effects of Spinally Administered Orphanin
FQ/Nociceptin
Although the seminal investigations of OFQ/N featured supraspinal administration of the peptide, opioids
play an equally crucial role in pain modulation in the
spinal level (see Yaksh, 1999). Although OFQ/N injected
intrathecally (10 nmol, i.t.) was initially reported to have
no effect on thermal nociception (Reinscheid et al.,
1995), a subsequent study reported a trend (p ⫽ 0.053)
toward enhanced morphine analgesia by intrathecal
MOGIL AND PASTERNAK
OFQ/N (Grisel et al., 1996) followed by additional support for spinal OFQ/N analgesia (Xu et al., 1996; King et
al., 1997). The situation has become more complicated
since then, as shown in Table 7. Strikingly low OFQ/N
doses spinally produce spontaneous pain, as evidenced
by caudally directed scratching, biting, and licking
(SBL) behaviors, and hypersensitivity to thermal and
mechanical stimuli. These SBL behaviors are reminiscent of those elicited by substance P and N-methyl-Daspartate (NMDA) and are eliminated by pretreatment
with morphine and neurokinin-1 (NK1) receptor antagonists, but not neurokinin-2 (NK2) or NMDA receptor
antagonists (Sakurada et al., 1999b). At higher OFQ/N
doses, a number of laboratories have observed analgesic
and anti-hyperalgesic/anti-allodynic effects. However,
some have been unable to demonstrate OFQ/N analgesia
at presumably effective doses. Still others have demonstrated anti-analgesic effects reminiscent of supraspinal
peptide, alone or in combination with hyperalgesia (see
Table 7).
Despite the many contradictions in the established
literature to date, most reviewers have concluded that
the dominant spinal action of high doses of OFQ/N is
inhibitory— congruent with the findings of all electrophysiological studies—producing behavioral analgesia
and/or anti-hyperalgesia/anti-allodynia (Henderson and
McKnight, 1997; Meunier, 1997; Harrison and Grandy,
2000; Xu et al., 2000). Wang and colleagues (1996) have
arrived at the same conclusion for the trigeminal system. Assuming that spinal OFQ/N is analgesic, the potential role of classical opioid receptors remains a further unresolved issue. Of the eight studies looking at the
effects of opioid antagonists on OFQ/N analgesia, only
two reported a reversal (King et al., 1997; Hao and
Ogawa, 1998). In another study, repeated administration of spinal OFQ/N resulted in the development of
tolerance to the peptide’s analgesic effects and crosstolerance to morphine (Jhamandas et al., 1998). This
finding, however, is directly contradicted by yet another
study that found no cross-tolerance (Hao et al., 1997).
Nociception-relevant elements in the spinal cord undergo anatomical and functional alterations after peripheral nerve injury or inflammation and this plasticity
is thought to be important in producing and maintaining
chronic pain states (Woolf, 1983). OFQ/N appears to be
no exception. OFQ/N levels and binding increase in the
dorsal horn of the spinal cord after inflammation (Jia et
al., 1998; Rosen et al., 2000). In one study, this increase
was bilateral, but restricted to the superficial laminae (I
and II) of the cord (Jia et al., 1998). Inflammation also
induces expression of the prepro-OFQ/N gene in the
dorsal root ganglion, although the increased synthesis of
OFQ/N was quite short-lived (⬍6 h) (Andoh et al., 1997).
In contrast to dynorphin, which was increased in the
dorsal horn after a Bennett model nerve injury, OFQ/N
levels in this study trended lower, although the decrease
did not achieve statistical significance. These findings
393
are hard to reconcile with data demonstrating that highdose OFQ/N⬘s depressive effect on the flexor reflex is
decreased in inflamed rats and increased somewhat in
nerve injured rats (Abdulla and Smith, 1998; also see
Hao et al., 1998; Xu et al., 1999a). This pattern of functional changes is exactly opposite to that of ␮ opioids,
which exhibit increased efficacy in inflammatory states
(Stanfa and Dickenson, 1995) and greatly reduced efficacy against neuropathic pain (Arner and Meyerson,
1988). As pointed out by Xu and colleagues (1999a),
however, the effectiveness of exogenously applied
OFQ/N is primarily determined by the status of NOP1
receptors, not endogenous peptide levels. No data have
thus far been collected as to whether NOP1 receptors are
altered after injury.
C. Effects of Peripherally Administered Orphanin FQ/
Nociceptin
In addition to their effects in the CNS, opioids can
produce analgesia in the periphery, especially in the
presence of inflammation (Stein et al., 1990; Kolesnikov
et al., 1996). This fact, along with the ability of OFQ/N to
affect transmitter release in the peripheral nervous system (see Giuliani et al., 2000), suggests that OFQ/N may
modulate nociception directly at the site of pain and/or
injury. A small number of studies have investigated this
possibility, again with somewhat conflicting results.
Two elegant studies by Inoue and colleagues (1998,
1999) demonstrated the ability of OFQ/N at remarkably
low doses, up to 10,000-fold lower than substance P and
1000-fold lower than bradykinin, to elicit the nociceptive
flexor reflex after intraplantar injection into the hindpaw. This effect appears to be secondary to local substance P release in the paw, since the phenomenon can
be blocked by inhibition of transmitter release by botulinum toxin, depletion of substance P by capsaicin, by
NK1 receptor antagonists, and is abolished in tachykinin-1 gene knockout mice (Inoue et al., 1998). In the
second study, however, a higher OFQ/N dose (1 nmol)
was analgesic, producing a complete blockade of substance P-induced flexor reflexes and SBL (Inoue et al.,
1999) (see Section VIII.D.5.). Another group, also using
higher doses, demonstrated the analgesic efficacy of
OFQ/N applied subcutaneously to the tail (Kolesnikov
and Pasternak, 1999). This analgesia was naloxone-reversible, but insensitive to antagonism by either ␮- or
␬-specific antagonists.
The modulatory effects of OFQ/N on rat knee joint
afferents were very recently studied by McDougall et al.
(2000). They found a sensitizing effect of OFQ/N in normal joints, and a desensitizing effect during hyper-rotation in acutely inflamed knees. Interestingly, both these
effects may be explained by the OFQ/N-substance P
interactions described above (Inoue et al., 1998; Lecci et
al., 2000b). However, Kumar and colleagues (1999) were
unable to demonstrate [3H]OFQ/N binding in human
synovial joint fluid or tissue. OFQ/N has been implicated
394
TABLE 7
Parameters and results of published studies investigating the effect on nociceptive sensitivity of spinally (i.t.)-injected OFQ/N
Effect
Species
Straina
Sex
Assayb
Controlc
Intensityd
Dosee
Reference
nmol, i.t.
Spontaneous nociception
Mouse
ddY
么
SBL
Veh
N.A.
3 ⫻ 10⫺9–3 ⫻ 10⫺5f
Inoue et al., 1999; Sakurada et al., 1999b, 2000
Mouse
ddY
么
Probe
Veh
N.A.
5.5 ⫻ 10⫺4–0.28f
Rat
Mouse
Mouse
Mouse
SD
ddY
ddY
ddY
乆
么
么
么
FR
HP
FR
HT
TWrh
None
Veh ⫹ BL
None
Veh
Veh ⫹ BL
N.A.
14 s
N.A.
9s
8s
⬍0.055
ⱖ2.75 ⫻ 10⫺9/kg
f
1 ⫻ 10⫺7–1
ⱖ1.5 ⫻ 10⫺6
ⱖ1.5 ⫻ 10⫺6
Okuda-Ashitaka et al., 1996; Hara et al., 1997;
Minami et al., 2000
Xu et al., 1996, 1999b
Hara et al., 1997, 2000; Minami et al., 1997
Inoue et al., 1999
None
BL
BL
BL
Veh ⫹ BL
Veh
Veh
N.A.
N.R.
2.5 s
4s
5s
5%, 50 ␮l
5%, 50 ␮l
ⱖ0.55
ⱖ0.55
ⱖ2.75
5.5
ⱖ3
ⱖ3.3
ⱖ1.7
Hyperalgesia/allodynia
Sakurada et al., 1999a,c
Analgesia
Rat
SD
么
Mouse
Rat
Rat
Rat
Rat
CD-1
SD
Wistar
SD
SD
么
么
么⫹乆
么
么
FR
TWrh
TWrh
TWrh
TWrh
F
F
Rat
Rat
Mouse
Mouse
Rat
Rat
Mouse
SD
SD
ICR
ICR
SD
SD
ddY
么⫹乆
么
么
么
N.R.
么
么
TWrh
F
F
TWrh
TWhw
F
F
Veh
Veh
Veh
BL
Veh ⫹ BL
Veh
Veh ⫹ NI
4s
5%, 50 ␮l
0.5%, 25 ␮l
6s
6s
5%, 150 ␮l
2%, 20 ␮l
ⱖ0.17
ⱖ5
ⱖ1
ⱖ1
0.55
ⱖ0.055
ⱖ0.17
Mouse
Mouse
Rat
Rat
NMRI
SW
SD
SD
么
么⫹乆
么
么
TWrh
TWhw
HP
HT
Veh
Veh ⫹ BL
Veh ⫹ BL
Veh ⫹ BL
7s
3.5 s
20 s
11 s
10
10
17
17
Rat
Mouse
Rat
Rat
Rat
SD
ICR
SD
SD
SD
么
么
么
么
么
VF
TWhw
TWhw
HP
VF
Veh
Veh ⫹ BL
Veh ⫹ BL
Veh ⫹ BL
None
60 g
N.R.
N.R.
N.R.
N.R.
17
0.055–3
10
10
10
Vanderah et al., 1998
Rat
SD
么
Rat
Mouse
SD
CD-1
乆
么
PP
TWrh
FS
TWrh
Veh
Veh
Veh
Veh
⫹
⫹
⫹
⫹
N.R.
2s
N.R.
3s
0.5–5
0.5–5
ⱖ1
0.0055
Jhamandas et al., 1998m
Dawson-Basoa and Gintzler, 1997n
Rady et al., 2001o
SD
SD
么
么
TWrh
TWrh
???
???
???
???
ⱖ0.055p
ⱖ0.055p
Zhu et al., 1998q
Zhang et al., 1997r
SD
SD
SD
么
么
么⫹乆
HT
VF
TWrh
VF
Veh ⫹ BL
Veh
Veh
Veh
11 s
50 g
4s
80 g
17
17
ⱖ0.55
ⱖ1.7
Yamamoto et al., 1997b, 2000as
Yamamoto and Sakashita, 1999bt
Xu et al., 1996
King et al., 1997
Hao et al., 1997
Tian et al., 1997a,b
Erb et al., 1997
Yamamoto et al., 1997a, 2000b; Yamamoto and
Sakashita, 1999a
Hao et al., 1998g
Hao and Ogawa, 1998
Kamei et al., 1999bh
Kamei et al., 1999ai
Wang et al., 1999cj
Wang et al., 1999a
Nakano et al., 2000k
No hyperalgesia or
analgesia
Reinscheid et al., 1995
Grisel et al., 1996l
Yamamoto et al., 1997a
Yamamoto and Nozaki-Taguchi, 1997;
Yamamoto et al., 2000a
Yamamoto and Sakashita, 1999b
Anti-analgesia
BL
BL
BL
BL
Hyperalgesia and antianalgesia
Rat
Rat
Anti-hyperalgesia/allodynia
Rat
Rat
Rat
Hao et al., 1998u
N.A., not applicable; N.R., not reported; ???, unknown.
a
Strain abbreviations: CD-1, Hsd:ICR; ddY, an inbred strain; ICR, Institute for Cancer Research (Swiss-derived); NMRI, a Swiss-derived European strain; SD,
Sprague-Dawley; SW, Swiss-Webster.
b
Assay abbreviations: HP, hot-plate test; HT, Hargreaves’ test of paw-withdrawal from radiant heat; F, formalin test; FR, spinal flexor reflex; FS, electric footshock test;
Probe, behavioral response to stroking on the flank with a paintbrush; PP, paw pressure (Randall-Selitto) test; SBL, caudally-directed scratching, biting, and licking behavior;
TWrh, radiant heat tail-withdrawal (tail-flick) test; TWhw, hot water tail-withdrawal test; VF, Von Frey test of mechanical sensitivity.
c
Control group abbreviations: All, all of the following were used; BL, comparison with baseline latencies; Veh, comparison with vehicle group; NI, comparison with
non-injected group.
d
For thermal assays, baseline latency or latency of control group is provided. For other assays, actual intensity parameters are listed.
e
Effective OFQ/N dose or dose range. Many of these studies investigated a wider overall dose range than that shown.
f
Biphasic dose-response relationship observed.
g
Analgesic effect was obtained in contralateral paws of spinally-injured, nerve-irradiated and inflamed animals.
h
In contrast to all other investigations in this section employing the formalin test, these investigators only observed an analgesic effect in the acute/early phase (0 –5 min)
of the biphasic formalin test.
i
In mice made diabetic with injections of streptozotocin, OFQ/N was even more potent, producing significant analgesia at a dose of 0.1 nmol.
j
In addition to a mild analgesic effect, i.t. OFQ/N produced an impressive potentiation of endomorphin-1 analgesia.
k
Hyperalgesic effects on tonic phase formalin licking were seen at a dose of 10 pg.
l
This study obtained an almost significant (p ⫽ 0.053) potentiation of spinal morphine analgesia by OFQ/N.
m
Anti-analgesic effect against systemic morphine analgesia. A dose of 10 nmol failed to block morphine analgesia and actually prolonged its duration. These investigators
also demonstrated OFQ/N analgesia in the TWrh and PP tests, but only at much higher doses (50 and 100 nmol).
n
Anti-analgesic effect against gestational and steroid-induced analgesia. In non-pregnant animals, no effect of OFQ/N on nociception was seen.
o
Anti-analgesic effect against spinal morphine analgesia.
p
Dose at which anti-analgesic effects were noted. Hyperalgesia was only seen at higher doses.
q
Anti-analgesic effect against fentanyl and U50,488 analgesia.
r
Anti-analgesic effect against electroacupuncture analgesia.
s
Blocked thermal hyperalgesia after a Bennett and Xie (1988) model neuropathic injury in one study, and after a Seltzer et al. (1990) model neuropathic injury in the
other. In a subsequent study, the finding in the Bennett model was weakly replicated, but the finding in the Seltzer model appeared not to be replicated.
t
A partial, and naloxone-sensitive blockade of mechanical allodynia from skin incision.
u
Blocked thermal hyperalgesia, mechanical allodynia, and cold allodynia after carrageenan inflammation, peripheral nerve injury, or ischemic spinal cord injury.
MOGIL AND PASTERNAK
in fibromyalgia where female sufferers display decreased plasma levels of the peptide (Anderberg et al.,
1998).
D. Reconciling the Literature
The preceding descriptions of OFQ/N effects on nociceptive phenomena at the supraspinal, spinal, and peripheral levels (see Tables 2 and 3) illustrate the considerable uncertainty that still surrounds the simplest of
questions: What are the actions of OFQ/N when injected? The next sections will address a number of factors that may be relevant to reconciling the divergent
results found in the literature and thus to illuminating
the endogenous role of OFQ/N.
1. Noxious Stimulus Modality. There is a large literature demonstrating differential processing of different
types of pain by neurochemically distinct circuitries (for
reviews, see Mogil et al., 1996c, 1999b). It is possible,
therefore, that activities of OFQ/N may be dependent
upon the nociceptive assay used. The majority of the
studies to date have used thermal assays (tail-flick/withdrawal, hot-plate tests) (Tables 2 and 3), which is to be
expected since these assays are easily performed and
commonly used in the opioid field. Supraspinal OFQ/N
anti-analgesia is a robust response and has been demonstrated against thermal, electrical, and chemical assays,
of varying durations (acute to chronic). Spinal hyperalgesia/allodynia and analgesia have been demonstrated
against thermal, chemical, and mechanical assays. Even
some of the less common findings, such as spinal antianalgesia or anti-hyperalgesia/anti-allodynia, have been
observed using multiple nociceptive assays. Overall,
there does not appear to be strong evidence at the
present time for modality-specific effects of the pronociceptive actions of OFQ/N.
OFQ/N analgesia, however, is less robust and far more
controversial and assay differences are more likely to be
important. Variations in the performance of specific
tests among laboratories and the tests themselves can
influence analgesic potency of opioids. However, it remains to be demonstrated whether assays play a major
role in the differences among reported observations.
2. Robustness of Various Phenomena. Not all of the
reported phenomena are equally robust. For example, of
the 16 studies reporting supraspinal hyperalgesia listed
in Table 6, half featured latency decreases, or formalin
rating increases, of ⬍40% compared with baseline
and/or vehicle values. By contrast, virtually all studies
reporting anti-opioid analgesic actions of OFQ/N demonstrated a complete blockade of even profound analgesia. Also, the supraspinal hyperalgesic actions of OFQ/N
are quite transient compared with the anti-analgesic
actions, with the former lasting only 15 to 30 min in
virtually all cases. One can easily point to degradation of
the peptide as an explanation of transient effects, but
such degradation does not prevent long-lasting antianalgesic actions (see especially Candeletti and Ferri,
395
2000). Particularly weak is the phenomenon of supraspinal OFQ/N analgesia defined as a quantal doubling of
the baseline tail-flick latency, which can be demonstrated in only 40% of CD-1 mice and was not seen in
two other strains (see Section VIII.D.3.) (Rossi et al.,
1996b, 1997). It should be noted that blockade of the
anti-opioid ␴1 receptor system with the ␴ receptor antagonist, haloperidol, dramatically enhanced the analgesic actions of OFQ/N and its fragments in all strains
tested (Rossi et al., 1997). Supraspinal OFQ/N analgesia
does seem to be more robust in the rat (Rossi et al.,
1998b).
Critically assessing the reliability or importance of
weak phenomena is difficult. Although they might represent chance occurrences, a finding may depend on a
particular set of organismic and parametric circumstances that would encourage replication within laboratories but not between them. Furthermore, small overall
effects may simply reflect the summation of opposing
processes, with one canceling out another (see Section
VIII.D.6.). Obviously, there is a need for additional attempts by independent laboratories to replicate and extend some of the OFQ/N phenomena reported.
3. Influence of Stress. The original investigations of
the supraspinal OFQ/N quantified the effect of the peptide relative to a control group receiving an isovolumetric injection of vehicle (Meunier et al., 1995; Reinscheid
et al., 1995). Although a reasonable control, it alone is
not sufficient since mice receiving intracerebroventricular injections are not at “baseline”. This is particularly
evident when dealing with nociceptive assays capable of
detecting stress analgesia. Employing either “no injection” and/or preinjection baseline control groups, depending on the nociceptive assay, Mogil and colleagues
(1996a) demonstrated that the apparent hyperalgesia
noted previously could be explained by the reversal of
stress-induced analgesia by OFQ/N. In these studies,
the apparent hyperalgesia compared with the vehicle
group was actually reversal of stress-induced analgesia
related to the injection when compared with the noinjection group (Mogil et al., 1996a).
Stress-induced analgesia is a well known, adaptive
phenomenon thought to represent the evolutionary impetus for the development of central pain inhibition
mechanisms, and thus the neural substrate on which
clinical analgesics like morphine act (Kelly, 1986). The
phenomenon can be produced by any number of environmental stressors and can be mediated by opioid or nonopioid neurochemistry (Lewis et al., 1980; Watkins and
Mayer, 1982). Although it is not widely appreciated that
procedures related to nociceptive testing can themselves
produce stress-induced analgesia, we have shown that
even intraperitoneal injections of saline can produce the
effect in some circumstances (Wilson et al., 1998), and
others have shown activation of the hypothalamic-pituitary-adrenal (HPA) axis and c-fos induction from this
mild stressor (Ryabinin et al., 1999). Although intrace-
396
rebroventricular injections performed by any number of
modifications of the method of Haley and McCormick
(1957) are often performed under light gaseous anesthesia, they may be considered a significant stressor in at
least three possible ways. First, the anesthesia itself
may be a stressful experience. Second, the injection proceeds directly through the skull at the coronal suture
and thus represents a mild trauma. Third, a nontrivial
volume of fluid is injected into the cerebral ventricles. In
the hands of Mogil and Grisel (Grisel et al., 1996; Mogil
et al., 1996a,b), this method of injection can produce
measurable analgesia against mild-to-moderate noxious
heat (up to 49°C). Intrathecal injections in the mouse
performed by the method of Hylden and Wilcox (1980)
are equally subject to the phenomenon (Grisel et al.,
1996; Mogil et al., 2000b). The probability of encountering this confound increases as the severity of the noxious
stimulus decreases. As the pain research community
increasingly switches its attention from analgesia to
mechanisms underlying hyperalgesia, increasingly mild
noxious stimuli are featured in experiments so that
“floor” effects can be avoided. Tables 2 and 3 document
the especially mild noxious stimulus parameters that
have been employed in the OFQ/N literature.
Although some have concluded that their prior data
may have been confounded in the manner described
above (Suaudeau et al., 1998), many laboratories have
continued to report supraspinal OFQ/N hyperalgesia,
even with the adoption of the recommended controls
(e.g., Calo’ et al., 1998). Although OFQ/N clearly can
reverse stress-induced analgesia, it also may have hyperalgesic activity in models with less of a confound.
Also, OFQ/N hyperalgesia has been reported on a number of occasions in the rat, a species in which i.c.v.
injections must proceed through an indwelling cannula,
minimizing the possibility of stress-induced analgesia
during the testing procedure itself. Therefore, other factors must be considered in an attempt to reconcile the
entire literature. Stress may, in fact, play a more general role here, especially given the demonstrated ability
of OFQ/N to act as an anxiolytic (Jenck et al., 1997;
Mamiya et al., 1998; Koster et al., 1999) (see Section
X.B.). Perhaps, then, the effect of exogenously administered OFQ/N is wholly dependent on the psychological
state of the subject at the time of testing, a state that can
be influenced by husbandry (e.g., fighting among grouphoused males), test-related stressors, and genetic factors
(see below). If so, then the considerable challenge of
parsing out such phenomena may yield large rewards in
our understanding of individual differences in pain sensitivity.
4. Organismic Factors: Species, Strain, and Sex Differences. An inspection of Tables 2 and 3 reveal that
species differences are not an obvious explanation of
discrepancies in this literature, as virtually all categories of OFQ/N effects include both mouse and rat studies. There are some notable exceptions, however. Spon-
taneous nociception and hyperalgesia/allodynia from
spinal OFQ/N is a phenomenon so far demonstrated only
in mice of the inbred ddY strain. Similarly, anti-hyperalgesia/anti-allodynia and anti-analgesia from spinal
OFQ/N has only been observed in the Sprague-Dawley
rat. There are only two examples to our knowledge of a
specific within-laboratory species comparison of OFQ/N
actions. Rossi and colleagues (1998b) noted that they
were unable to demonstrate the supraspinal OFQ/N hyperalgesia in the rat that they had observed repeatedly
in the mouse. The systematic examination of Vanderah
et al. (1998) of OFQ/N actions used both rats and mice,
although they detected no reliable effects of OFQ/N on
nociception in either species. It should also be noted that
OFQ/N has been administered to a nonmammalian species, the land snail (Cepaea nemoralis), and found (with
all appropriate controls) to produce hyperalgesia on the
hot-plate test (Kavaliers and Perrot-Sinal, 1996; Kavaliers et al., 1997). Species differences between binding
and coupling properties of the mouse versus human
NOP1 receptor have demonstrated, and the authors suggested that this, in combination with possible species
differences in receptor reserve, may account for some of
the contradictions in the established literature (Burnside et al., 2000).
We believe that intraspecies genotypic differences
(i.e., strain differences) may be more useful as an explanation of some of the inconsistencies seen thus far. As
noted above, Rossi and colleagues (1997) observed a
modest analgesia from supraspinal OFQ/N in CD-1 mice
that was not seen in outbred Swiss-Webster or inbred
BALB/cJ mice. This finding inspired Mogil et al. (1999a)
to examine the effects of supraspinal OFQ/N, and supraspinal injections themselves, on thermal nociception
in six mouse strains: outbred CD-1 and Swiss-Webster
mice, and inbred AKR/J, BALB/cJ, C3H/HeJ, and CBA/J
mice. Intracerebroventricular injections per se produced
significant increases in 47°C tail-withdrawal latency at
15-min postinjection in four of six strains. This strain
dependence is not entirely surprising, since strain-dependent activation of the HPA axis following systemic
needle injection has been demonstrated (Ryabinin et al.,
1999). OFQ/N reversed the injection-related analgesia
in two of the strains, Swiss-Webster and BALB/cJ. In no
strain, however, was statistically significant hyperalgesia or analgesia observed, although a strong trend toward the latter was obtained in CBA/J mice (Mogil et al.,
1999a). These findings may help explain why only some
investigators have observed the phenomenon of vehicle
injection stress-induced analgesia in the mouse (e.g.,
Mogil et al., 1996a; Suaudeau et al., 1998), whereas
others have not (Rossi et al., 1997; e.g., Calo’ et al.,
1998). Also of interest is the fact that strain differences
in OFQ/N immunoreactivity have been demonstrated
between DBA/2J and C57BL/J mice (Ploj et al., 2000),
two strains with highly divergent nociceptive and analgesic sensitivities (see Mogil et al., 1996d; Mogil, 1999).
MOGIL AND PASTERNAK
However, those differences are found in the frontal cortex and hippocampus only (C57BL/6J ⬎ DBA/2J), and
thus it is difficult to see their direct relevance to acute
nociception.
Evidence for important sex differences in the mediation and opioid modulation of nociception continues to
mount (Berkley, 1997; Kest et al., 2000; Mogil et al.,
2000a). However, sexually dimorphic OFQ/N functioning is unlikely to explain contradictions in the existing
literature, as this literature has overwhelmingly employed male subjects (see Tables 2 and 3). Furthermore,
in those few studies that have tested both sexes (Grisel
et al., 1996; Hao et al., 1998; Mogil et al., 1996a,b, 1999a;
Tian et al., 1997a,b), no sex differences in the effects of
OFQ/N were reported.
5. Dose Dependence. The OFQ/N dose employed may
have a dramatic impact on the effect of the peptide. With
respect to supraspinal OFQ/N, both anti-analgesic and
hyperalgesic effects have been observed over broad dose
ranges (125,000- and 2,000-fold, respectively). By contrast, analgesia has only been observed at high doses
(ⱖ5.5 nmol). In many of the studies where anti-analgesia and hyperalgesia were observed, the former was
obtained with lower doses than the latter (Zhang et al.,
1997; Zhu et al., 1997, 1998; Wang et al., 1999a,c; Citterio et al., 2000).
Spinal and peripheral OFQ/N reveals an even more
obvious dose dependence. Extremely low doses (attomolar to picomolar range) of the peptide produce SBL behaviors and hyperalgesia/allodynia, whereas higher
doses (picomolar to nanomolar) produce analgesia. In
the studies of Xu and colleagues (1996, 1999b), the facilitation of the flexor reflex by low doses of OFQ/N was
weak, brief, and unreliable, whereas the inhibition of the
reflex caused by higher doses was prolonged and robust.
The studies of the Japanese groups, however, have consistently documented inverted U-shaped dose-response
relationships between OFQ/N and pronociceptive outcomes, with SBL behaviors, flexor reflex facilitation, and
thermal hyperalgesia peaking in the low femtomolar
range (Hara et al., 1997; Inoue et al., 1999; Sakurada et
al., 1999b). This bell-shaped dose-response curve contrasts with that of other pain-producing peptides, such
as substance P. However, Inoue and colleagues (1999)
explain the biphasic action of OFQ/N entirely in terms of
substance P. At very low doses, OFQ/N is likely stimulating nociceptive primary afferents containing substance P, causing the release of the latter peptide. At
higher doses, OFQ/N is still causing the release of substance P but now is able to activate spinal NOP1 receptors (via Gi/o). The activation of NOP1 receptors in the
spinal cord produces analgesia via the inhibition of
postsynaptic substance P-mediated actions (Inoue et al.,
1999). Ito’s laboratory has used dose relationships to
dissociate mechanisms underlying OFQ/N mechanical
allodynia (seen only from 0.55 pmol to 0.28 nmol) and
OFQ/N thermal hyperalgesia (seen at all doses tested
397
above 2.75 amol) (Okuda-Ashitaka et al., 1996; Hara et
al., 1997). Both phenomena are inhibited by morphine
and neonatal capsaicin treatment and mediated by glycine receptors (Hara et al., 1997; Minami et al., 2000).
However, only the allodynia is sensitive to blockade by
antagonists of the glutamate receptor-nitric oxide pathway and by prostaglandin D2 (PGD2) (Hara et al., 1997;
Minami et al., 1997, 2000). In contrast, only the OFQ/N
hyperalgesia is mediated by substance P (Minami et al.,
2000). This same dissociation between allodynia and
hyperalgesia was demonstrated by this group for prostaglandin E2 (PGE2) (Minami et al., 1996).
A dose-dependent relationship exists between OFQ/N
and release of the endogenous opioid, enkephalin, in the
guinea pig myenteric plexus (Gintzler et al., 1997). This
in vitro preparation contains strikingly similar proportions of ␮, ␬, and ␦ receptors compared with the CNS
(Gyang et al., 1964), and has been used to investigate
the release of enkephalin by ␮-receptor agonists (Glass
et al., 1986). With blockade of classical opioid receptors
with naloxone, OFQ/N at low concentrations (1–10 nM)
inhibited the electrically stimulated release of met-enkephalin by 40%. At higher concentrations (100 –1000
nM), OFQ/N facilitated enkephalin release by up to 50%
(Gintzler et al., 1997). Note that this biphasic pattern is
exactly the opposite of that characterizing the ␮-opioid
agonist, sufentanil, which facilitates enkephalin release
at low doses and inhibits it at higher doses (Xu et al.,
1989). This dose-dependent pattern of enkephalin release could be invoked to explain inconsistencies in both
the supraspinal and spinal compartments, since enkephalin plays an important role in nociceptive processing at both levels (Millan, 1986). The decreased enkephalin release produced by low concentrations of
OFQ/N would tend to be pronociceptive, perhaps partially accounting for the hyperalgesia/allodynia seen after low dose spinal injections and the anti-analgesia/
anti-hyperalgesia seen after supraspinal injections. The
increased enkephalin release produced by high concentrations of OFQ/N would tend to be antinociceptive, perhaps accounting for the high dose spinal analgesia seen
by many and/or the supraspinal analgesia seen by Pasternak’s laboratory. These possibilities are purely speculative, of course, since OFQ/N has not yet been shown
to release enkephalin in either the spinal cord or the
brain. Most intriguing is the fact that while OFQ/N was
shown to modulate the evoked release of enkephalin, it
did not affect the basal release of the peptide, prompting
the authors to conclude that “the hyperalgesic actions of
centrally administered nociceptin should be expected to
be particularly robust when pain thresholds are elevated
due, in part, to augmented enkephalin neurotransmission” (Gintzler et al., 1997). One way of elevating pain
thresholds via augmented enkephalin neurotransmission is to expose animals to environmental stressors
(e.g., Christie et al., 1981; Schmidt et al., 1991; but see
Konig et al., 1996; Mizoguchi et al., 1997). The blockade
398
of enkephalin release by OFQ/N may thus provide a
mechanistic explanation for the behavioral hyperalgesia
produced by supraspinal OFQ/N in situations involving
stress-induced analgesia (test-related or otherwise).
A final issue related to dose is the possibility that the
exogenous administration of OFQ/N at different doses
may lead to the differential production of metabolites.
Such metabolites may be bioactive and may functionally
interact with OFQ/N, changing its apparent effect. This
has been explicitly demonstrated by Sakurada et al.
(2000), who observed that N-terminal fragments of
OFQ/N, specifically OFQ/N(1–7), OFQ/N(1–9), and
OFQ/N(1–13), fully blocked the SBL behaviors induced
by low OFQ/N doses. OFQ/N(1–13) was actively antagonistic at doses equimolar to OFQ/N, suggesting an endogenous role. In a separate study by the same group,
OFQ/N(1–7) was shown to block supraspinal OFQ/N
hyperalgesia but not spinal OFQ/N analgesia (Sakurada
et al., 1999c). Supraspinal OFQ/N(1–7) and OFQ/N(1–
11) produce analgesia in mice without evidence of hyperalgesia (Rossi et al., 1997) and these fragments were
unable to reverse morphine analgesia (King et al., 1998).
Finally, the hexapeptide OFQ/N(1– 6) exhibits a biphasic pattern of responses on the hot-plate test, but not the
hot water tail-withdrawal test, whereby a short-lived
analgesia was replaced by a modest hyperalgesia (Suder
et al., 1999). Intriguingly, this pattern is entirely opposite of the pattern observed by Rossi and colleagues
(1997) using the full peptide. The hyperalgesia but not
the analgesia was reversed by noncompetitive antagonists at the NMDA receptor: MK-801, a pore blocker,
and L-701,324, an allosteric glycine site blocker (Suder
et al., 1999). Experiments in the land snail also implicate a role for NMDA receptors in OFQ/N hyperalgesia
(Kavaliers et al., 1997).
OFQ/N fragments appear to have poor affinity for
NOP1 (Dooley and Houghten, 1996; Mathis et al., 1997).
Even though OFQ/N(1–11) labels NOP1 sites modestly
well (Ki ⬃50 nM), its affinity does not compare to OFQ/N
(KD ⬃50 pM), so competitive antagonism of NOP1 receptors is unlikely. These peptides do affect cAMP accumulation, however, suggesting the existence of functionally
heterogeneous receptors related to NOP1, possibly via
alternative splicing (see Section VI.) (Mathis et al.,
1997). Given that the vast majority of radiolabeled
[Tyr14]OFQ/N administered supraspinally is metabolized within 15 min, it is possible that OFQ/N analgesia,
which exhibits a delayed onset, may be due entirely to
its conversion to bioactive metabolites (Rossi et al., 1997;
Suder et al., 1999).
6. Opioid Tone. A recent study by Lutfy and Maidment (2000) demonstrated that a hyperalgesic effect of
high doses of OFQ/N (15 or 30 nmol) could be revealed by
pharmacological blockade of ␮ receptors by naloxone or
CTOP. These investigators, using the hot-plate test, observed no effect on nociception of OFQ/N unless ␮ receptors were previously blocked by ␮ antagonists. They
argue that in addition to OFQ/N exerting an anti-opioid
effect, ␮ receptors might be regarded as anti-OFQ/N,
counteracting a pronociceptive action of OFQ/N. That is,
the failure to observe OFQ/N hyperalgesia is due to a
counteracting opioid “tone”, and not to stress-induced
analgesia. The tone could not exist prior to the OFQ/N
injection, or else naloxone pretreatment would have produced an apparent hyperalgesia, which was not seen
(Lutfy and Maidment, 2000). Therefore, the OFQ/N injection must have released classical endogenous opioids
acting at ␮ receptors. Since there was no evidence in
their study of injection-related analgesia, one must conclude that OFQ/N itself caused the release of the opioid,
as has been shown to be the case in the guinea pig
myenteric plexus at high OFQ/N concentrations (Gintzler et al., 1997) (see Section VIII.D.5.). OFQ/N has also
been shown to cause the release of stress hormones
(ACTH and corticosterone) after supraspinal injection
(Devine et al., 2001), suggesting another mechanism by
which endogenous opioids may be indirectly released. If
this result is replicated, it may represent a major advance in our understanding of OFQ/N actions. It still
remains unclear, however, how i.c.v. OFQ/N could activate a ␮ receptor-mediated analgesia and simultaneously exert anti-analgesic actions at the same doses.
This observation may be consistent with the earlier report showing both an opioid antagonist-sensitive OFQ/N
analgesia, perhaps mediated by the release of endogenous opioids, and hyperalgesia (Rossi et al., 1997).
E. Effects of Other NOP1 Receptor Agonists
The actions of a number of peptidergic ligands other
than OFQ/N with affinity for the NOP1 receptor have
been examined to shed light on the role of the system in
nociception. For example, the amide form of OFQ/N,
NCNH2, binds to NOP1 receptors with equal or greater
affinity than the natural peptide (Calo’ et al., 2000a).
Calo’ et al. (1998) observed an identical and equimolar
hyperalgesic and anti-analgesic effect of supraspinal
NCNH2 compared with OFQ/N on the hot water tailwithdrawal assay. Bertorelli et al. (1999) observed supraspinal NCNH2, but not OFQ/N, hyperalgesia in a rat
arthritis model using complete Freund’s adjuvant on the
test of Hargreaves et al. (1988) in both the arthritic and
the contralateral hindpaw. This finding points out the
utility of using stabilized derivatives with reduced susceptibility to peptidases, like NCNH2, as functional
probes (Calo’ et al., 2000a). NC(1–13)NH2 is another
amidated fragment retaining full agonist potency when
injected supraspinally. In contrast, NC(1–9)NH2 was
entirely without effect (Calo’ et al., 1998). The hexapeptide ac-RYYRWK-NH2, identified from a combinatorial
library (Dooley et al., 1997), displays full agonist properties in behavioral assays (Berger et al., 2000), but so
far its effect on nociception has not been reported.
Ro 64 – 6198 [(1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1Hphenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-
MOGIL AND PASTERNAK
one] is a nonpeptidergic NOP1 receptor agonist with full
activity. It is particularly appealing since it can be used
systemically due to its ability to traverse the blood-brain
barrier. Despite its OFQ/N-like anxiolytic effects (see X.B),
Ro 64–6198 did not alter thermal or mechanical nociceptive thresholds in the same dose range (Jenck et al., 2000).
Its effects on other nociception-related phenomena have
not yet been tested. Finally, in vitro data raise the possibility that the clinically important opiate, buprenorphine
(Temgesic), is a partial agonist of NOP1 receptors (BlomsFunke et al., 2000). Although its lack of selectivity among
the opioid and NOP1 receptors precludes its use as a research tool to explore the pharmacology of the NOP1 receptors, buprenorphine may have interactions with NOP1
receptors that may be of considerable value in explaining
the complexities of buprenorphine’s pharmacology, such as
biphasic or even triphasic dose-response curves and antiopioid activity (Dum and Herz, 1981; Pick et al., 1997).
F. Phenotypes of Knockout Mice
The absence of a NOP1 receptor antagonist until recently, led researchers in the OFQ/N field to a useful
alternative to pharmacology, gene deletion. Transgenic
“knockout” mice lacking functional expression of the
NOP1 receptor gene (Oprl1; chromosome 2, 110 cM) and
the ppOFQ/N gene (Npnc1; genomic location unknown)
have been constructed and tested for pain-related phenotypes.
The NOP1 receptor mutants were developed first, and
as expected, were insensitive to OFQ/N. Neither supraspinal OFQ/N hyperalgesia nor spinal OFQ/N flexor
reflex facilitation were found in the knockouts (Nishi et
al., 1997; Noda et al., 1998; Ueda et al., 2000). Far more
important, however, was the attempt to determine
whether basal nociceptive thresholds or analgesic sensitivity to opioids were altered in these animals. The answer in both cases appears to be no; knockout mice are
equivalently sensitive to their wildtype counterparts on
the thermal tail-flick and hot-plate tests, the mechanical
tail-clip test, the electric foot-shock test and the acetic
acid abdominal constriction test (Mamiya et al., 1998;
Nishi et al., 1997; Ueda et al., 1997; 2000). In addition,
morphine’s analgesic potency was unchanged in knockout animals following systemic injection over a range of
doses (Nishi et al., 1997; Noda et al., 1998; Ueda et al.,
1997; 2000). Knockout studies of this type (see Mogil and
Grisel, 1998 for review), can be very useful, but always
face the potential problem of compensation by other
genes. In these NOP1 receptor knockout studies, the
authors concluded that the lack of differences between
wildtype and knockout mice indicates that NOP1 receptors are not essential for the determination of nociceptive threshold (Nishi et al., 1997). This implies the absence of a “basal tone” (see Discussion in sections
VIII.D.3 and VIII.D.5 above), much like the enkephalins
have little “basal tone”, as shown by the limited actions
of naloxone in naı¨ve animals. This leaves the important
399
question of the status of stress-induced analgesia in
these mutants.
Two separate groups have generated mice lacking the
gene for the precursor of OFQ/N, ppOFQ/N. Koster and
colleagues (1999) reported a decreased basal sensitivity
on the radiant heat tail-withdrawal (i.e., classic tailflick) test in the knockout animals. This difference between the ppOFQ/N and the NOP1 knockout mice might
reflect differences between the models: a) by eliminating
ppOFQ/N, all the peptides within the precursor also are
lost, including nocistatin and OFQ2/NocII peptides; b)
receptors other than NOP1 may mediate the effects of
OFQ/N and related peptides; and c) different embryonic
stem cell lines were used in the two projects, with concomitantly different genetic backgrounds (Simpson et
al., 1997; see Mogil and Grisel, 1998). A fascinating
aspect of this study is restriction of the decreased sensitivity of ppOFQ/N mutants to male mice that were
group housed; isolated knockout male mice were equivalently sensitive to isolated wildtypes. This is purported
to be a stress-related effect, since knockouts were found
to be diminished in their ability to adapt to stress (Koster et al., 1999) (see Section X.B.). Essentially, the
authors argue that mice lacking OFQ/N and other products of the ppOFQ/N gene are tonically stressed, and thus
exhibiting tonic stress-induced analgesia. The implication,
therefore, is that OFQ/N may serve endogenously to ameliorate stress, and thus stress-induced analgesia.
Although this is a very attractive and powerful hypothesis, its implications for understanding OFQ/N⬘s
effects on nociception are complicated by findings from
Pintar’s laboratory, in which independently derived
ppOFQ/N knockouts exhibited an increased sensitivity
in the hot water tail-withdrawal test (Chen et al., 1999).
The contrasting phenotypes are not due to any differences between the closely related radiant heat and hot
water versions of the assay, since the increased sensitivity of the Pintar mutants is seen in both versions
(J. S. Mogil, unpublished data).
G. Effects of NOP1 Down-Regulation or Blockade
Another major approach to studying the role of OFQ/N
and NOP1 receptors in nociception has been to downregulate available receptor sites with antisense treatment or block them acutely with antagonists. This strategy has a number of advantages compared with the
exogenous administration of drugs in interpreting the
constitutive role of OFQ/N.
1. Antisense Studies. Antisense studies can be used
alone to explore the tonic activity of OFQ/N systems or
in conjunction with a drug to confirm the specificity of
the drug’s actions. In the opioid field, investigators typically have used short oligodeoxynucleotide probes consisting of approximately twenty bases targeting a region
of the mRNA near the translational start site. However,
antisense probes can effectively down-regulate proteins
virtually anywhere along the mRNA, provided an appro-
400
priate sequence is available (Kolesnikov et al., 1997;
Pasternak and Pan, 2000; Standifer et al., 1994). This
ability to target individual exons has led to the concept
of antisense mapping, which has proven valuable in the
elucidation of the role of alternative splicing (Pasternak
and Standifer, 1995; Kolesnikov et al., 1997).
Earlier, the role of antisense mapping in classifying
the NOP1 receptor was briefly discussed (see Section
IV.). The initial cloning studies with the NOP1 receptor
pointed out the poor affinity of traditional opioids for
this site and raised questions regarding its pharmacological significance. In these initial antisense studies
against NOP1, six different antisense probes targeting
the second and third coding exons all blocked the analgesic actions of naloxone benzoylhydrazone, strongly implying a role of NOP1 in ␬3 analgesia (Pan et al.,. 1994;
1995). However, the inactivity of antisense against the
first coding exon, as well as other factors, also pointed
out that NOP1 was not the same as the ␬3 opioid receptor. Thus, the use of antisense approaches must be interpreted cautiously. Some probes may be active while
others based upon a different exon of the same gene are
not. Although subtle, these issues must always be considered when assessing antisense paradigms. Optimally, each exon should be individually targeted, but
this is not always feasible.
Antisense mapping NOP1 revealed interesting patterns for OFQ/N analgesia and hyperalgesia (Rossi et
al., 1997). Antisense targeting exon 1 blocks OFQ/N
hyperalgesia in the radiant heat tailflick assay, as well
as its anti-opioid actions, while antisense oligodeoxynucleotides targeting the second and third coding
exons are inactive (Rossi et al., 1997; King et al., 1998).
Zhu and colleagues (1996; 1997) also demonstrated the
utility of a NOP1 antisense against the first coding exon
in blocking the anti-opioid effect of OFQ/N on morphine
and electroacupuncture analgesia. Conversely, antisense based upon the first coding exon of NOP1 was
inactive against OFQ/N analgesia, while probes targeting the second and third coding exons effectively blocked
OFQ/N analgesia. Thus, these mapping studies imply
that both OFQ/N analgesia, hyperalgesia and anti-opioid actions are mediated through receptors generated by
the gene producing NOP1 receptors. Yet, it appears that
the receptors responsible for analgesia are distinct from
those important in hyperalgesia and anti-opioid actions.
One of the seminal OFQ/N papers demonstrated that
an antisense oligonucleotide against the first coding
exon of the murine NOP1 clone injected daily for four
days produced analgesia on the hot-plate test while a
missense control was inactive (Meunier et al., 1995).
Zhu and colleagues (1996; 1997) replicated this finding
on the tail-shock and formalin tests, and also demonstrated the utility of NOP1 antisense in blocking the
anti-opioid effect of OFQ/N on morphine and electroacupuncture analgesia.
Finally, in the first study looking at the effects of
antisense directed toward ppOFQ/N mRNA, Candeletti
and Ferri (2000) demonstrated that repeated intracerebroventricular antisense treatment potentiated morphine analgesia, supporting an anti-opioid role of the
peptide.
2. Pharmacological Antagonists. Both transgenic
knockout and antisense approaches have limitations
(see, e.g., Mogil and McCarson, 2000), and thus the
search for a selective and competitive NOP1 antagonist
has been one of the major priorities in this field since
1996. One group found that ␴ receptor ligands (e.g.,
carbetapentane, rimcazole) blocked inward potassium
currents induced by OFQ/N acting on recombinant
NOP1 receptors (Kobayashi et al., 1997), but these compounds are nonselective and of low potency. The ␬3 ligand, NalBzoH, has also been proposed as a competitive
NOP1 antagonist based on data obtained from transgenic receptor knockouts and one group’s success in
blocking OFQ/N hyperalgesia, hypolocomotion, and
memory impairment with the compound (Noda et al.,
1998; Mamiya et al., 1999). However, we have collected
data suggesting that NalBzoH does not block supraspinal OFQ/N⬘s anti-analgesic actions but rather enhances
them (J. S. Mogil and J. E. Grisel, unpublished data) and
in biochemical assays NalBzoH has at least partial agonist activity in cyclase assays with the cloned murine
NOP1 receptor (Pan et al., 1994, 1995). The dextrorotatory enantiomer of the nonpeptidic ␦-opioid ligand, TAN67, blocks spinal OFQ/N analgesia in two different assays, but this antagonism was probably not competitive
(Kamei et al., 1999b). The peptide retro-nociceptin methylester also noncompetitively antagonized OFQ/N in the
in vitro guinea pig ileum assay and elicited a naloxoneinsensitive analgesia on the tail-pinch test, although it
did not affect OFQ/N hyperalgesia (Jinsmaa et al.,
2000).
Considerable excitement surrounded the publication
of the properties of the pseudopeptide [Phe1␺(CH2-NH)
Gly2]-nociceptin(1–13)-NH2, which selectively antagonized NOP1 receptors in the guinea pig ileum and mouse
vas deferens assays (Guerrini et al., 1998). Subsequent
studies showed, however, that [Phe1␺(CH2-NH)Gly2]nociceptin(1–13)-NH2 is a partial agonist with low efficacy, explaining its actions as an antagonist, partial
agonist, or even full agonist depending on the assay. In
vitro, the peptide is an antagonist in cells expressing low
levels of NOP1 receptors and is a partial or full agonist
in cells expressing high levels of the receptor (Burnside
et al., 2000). In most in vivo assays and all nociceptive
tests the peptide shows full agonist properties, producing supraspinal hyperalgesia (Calo’ et al., 1998; Bertorelli et al., 1999; Wang et al., 1999b; Candeletti et al.,
2000), anti-opioid effects (Calo’ et al., 1998; Grisel et al.,
1998), and intrathecal analgesia (Xu et al., 1998; Wang
et al., 1999b; Candeletti et al., 2000). In an electrophysiological study of spinal cord dorsal horn neurons,
MOGIL AND PASTERNAK
[Phe ␺(CH2-NH)Gly ]-nociceptin(1–13)-NH2 inhibited
C-fiber evoked responses just like OFQ/N (Carpenter
and Dickenson, 1998). Unlike the endogenous peptide,
however, the effect was partially naloxone-reversible,
prompting the authors to suggest that [Phe1␺(CH2-NH)Gly2]-nociceptin(1–13)-NH2 may have some activity at
spinal ␮ or ␦ receptors as well.
The next year, the same Italian group identified another
peptide in a structure-activity study, [Nphe1]nociceptin(1–
13)NH2, with uniformly antagonistic properties in all assays thus far examined (Calo’ et al., 2000b; Polidori et al.,
2000a; Rizzi et al., 2000). Not only is [Nphe1]nociceptin(1–
13)-NH2 fully able to reverse the supraspinal hyperalgesic
and anti-opioid actions of OFQ/N, but the antagonist can
potentiate morphine analgesia (Calo’ et al., 2000b; Rizzi et
al., 2000). This corresponds to the original prediction by
Mogil and colleagues (1996a) regarding the effect of an
antagonist once developed, and presages a clinical role for
such drugs as adjuncts to opiate pharmacotherapy. Interestingly, whereas [Nphe1]nociceptin(1–13)-NH2 very efficaciously potentiated the analgesic effects of morphine
given supraspinally, it was only marginally effective upon
systemic morphine analgesia unless animals were previously rendered tolerant to morphine (Rizzi et al., 2000).
This finding supports the notion that OFQ/N⬘s anti-opioid
actions are exerted supraspinally and also contributes to
the evidence implicating OFQ/N in the expression of morphine tolerance (see Section X.C.). Less clear is the critical
issue of whether [Nphe1]nociceptin(1–13)-NH2 can produce analgesia per se. In one study, using the moderately
noxious 48°C hot water tail-withdrawal test, a significant
(up to 150% increase in latencies), dose-dependent (3–30
nmol), but relatively short-lasting, analgesic effect was
demonstrated (Calo’ et al., 2000b). In another study, using
the highly noxious 55°C test, no analgesia could be elicited
from a 30-nmol dose of the antagonist (Rizzi et al., 2000).
Two nonpeptidic NOP1 antagonists were developed and
tested for their effects on nociceptive responses. JTC-801
[N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride], having at least 12.5-fold
selectivity over other opioid receptor types, blocked spinal
OFQ/N-induced allodynia and produced naloxone-insensitive, but not dose-dependent, analgesia in the 55°C hotplate and formalin tests (Shinkai et al., 2000). Another
compound, J-113397 [1-[(3R,4R)-1-cyclooctylmethyl-3hydroxymethol-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] showed much higher selectivity (⬎350fold) over classical opioid receptor types (Ozaki et al.,
2000a,b). Injected subcutaneously in mice, which minimizes the potential of stress effects, J-113397 dose dependently reversed supraspinal OFQ/N hyperalgesia in the
radiant heat tail-withdrawal test (Ozaki et al., 2000a).
However, even though minimally noxious parameters
were used, as illustrated by the relatively long baseline
latencies of approximately 9 s, no analgesia from this compound was detected at doses up to 30 mg/kg.
1
2
401
Thus, the question of whether tonic activation of
NOP1 receptors by OFQ/N contributes to a nociceptive
threshold-controlling tone remains unanswered, as summarized in Table 8, despite the unique advantages of
transgenic knockouts, antisense, and especially pharmacological antagonist models. For now, at least, the
mystery of the true endogenous role of OFQ/N in nociception has failed to yield to simple explanations and
technological advances.
H. Mechanisms of Orphanin FQ/Nociceptin Actions:
Ubiquitous Cellular Inhibition as a Unifying
Hypothesis?
Wide interest in this novel neurotransmitter was engendered by the seeming incongruity between its cellular actions that mimicked classical opioids and its behavioral effects, at least supraspinally, that opposed
those of classical opioids. Just like ␮, ␬, and ␦ receptors,
activation of NOP1 receptors by OFQ/N is associated
with inhibition of cAMP formation (via Gi/Go-mediated
intracellular signaling), closure of voltage-gated N-type
calcium channels, enhancement of an inwardly rectifying potassium conductance, and ultimately, reduction of
neuronal excitability (i.e., cellular inhibition) (Hawes et
al., 2000). The cellular neurophysiological actions of
OFQ/N are also identical to those of classical opioid
agonists, including the inhibition of transmitter release
in the spinal cord (Moran et al., 2000). Such properties
are what led both original investigations to search for
behavioral analgesia from OFQ/N; it was considered a
paradox that they demonstrated supraspinal hyperalgesia instead. Of course, this is only paradoxical if one fails
to consider the potential of interactions at the level of
neural circuits. This type of analysis has been used
successfully, for example, to explain the anti-␮-opioid
actions of ␬-opioid agonists like dynorphin (Pan et al.,
1997). It is to such a systems-level hypothesis that we
now turn, although it should be noted that the heterologous desensitization of ␮-opioid receptors by NOP1 activation in CHO cells (Hawes et al., 1998) might in
theory provide a purely cellular explanation of OFQ/N⬘s
anti-opioid actions. This is somewhat unlikely to be relevant in vivo, however, since ␮ and NOP1 receptors are
not colocalized in nociception-relevant loci (Schulz et al.,
1996; Monteillet-Agius et al., 1998).
Heinricher and colleagues (1997) investigated the
electrophysiological and behavioral effects of OFQ/N in
the nucleus raphe´ magnus of the RVM of lightly anesthetized rats. The RVM is a brain stem locus critical for
analgesia; it receives afferent input from the PAG and
sends a nociception-modulating outflow to the dorsal
horn of the spinal cord (Basbaum and Fields, 1984;
Fields et al., 1991). Previous work has demonstrated the
existence of three types of neurons in the RVM: ON cells,
which fire immediately prior to the occurrence of nociceptive reflexes (i.e., tail-withdrawals from noxious
heat); OFF cells, which pause their firing immediately
402
TABLE 8
Evidence for and against a role for the OFQ/N/NOP1 system in the
tonic control of nociceptive thresholds under basal conditions
Yes
1) I.c.v. administration of antisense oligonucleotides directed at
NOP1 mRNA is analgesic (Meunier et al., 1995; Zhu et al.,
1996, 1997; Rossi et al., 1997).
2) I.c.v. administration of the antagonist [Nphe1]nociceptin(1–
13)NH2 is analgesic on the 48°C hot water tail-withdrawal
test (Calo’ et al., 2000b).
3) Intracerebroventricular administration of the antagonist JTC801 is analgesic (Shinkai et al., 2000).
4) Koster and colleagues’ (1999) ppOFQ/N knockouts display
increased baseline tail-withdrawal latencies.
No
1) NOP1 transgenic knockouts do not display altered nociceptive
thresholds (Nishi et al., 1997; Ueda et al., 1997, 2000; Noda
et al., 1998).
2) Intracerebroventricular administration of the antagonist
[Nphe1]nociceptin(1–13)-NH2 is not analgesic on the 55°C hot
water tail-withdrawal test (Calo’ et al., 2000b).
3) Intracerebroventricular administration of the antagonist J113397 is not analgesic (Ozaki et al., 2000a).
4) Chen and colleagues’ (1999) ppOFQ/N knockouts display
decreased baseline tail-withdrawal latencies (see above).
prior to nociceptive reflexes; and NEUTRAL cells, which
do not change their firing (see Fields et al., 1991). Opioid
analgesia is mediated by the direct inhibition of ON cells
by the opioids that then disinhibit (i.e., excite) OFF cells,
whose firing prevents the tail-withdrawal reflex (Heinricher et al., 1994). Thus, an anti-opioid action of OFQ/N
could result by the peptide either: a) preventing classical
opioid inhibition of ON cells, or b) directly inhibiting
OFF cells.
Not surprisingly, given previous in vitro data (Connor
et al., 1996; Vaughan and Christie, 1996; Vaughan et al.,
1997), OFQ/N profoundly suppressed the firing of all
three cell classes in the RVM (Heinricher et al., 1997).
Iontophoretically applied OFQ/N blocked the inhibition
of tail-withdrawal reflexes by DAMGO applied to the
same site, an anti-opioid action, but had no effect when
applied alone. The parsimonious explanation of these
findings is that OFQ/N suppressed OFF cell firing, preventing opioid disinhibition from activating those same
neurons to produce analgesia (Heinricher et al., 1997).
ON cell firing would have been inhibited by both OFQ/N
and DAMGO, with no net change in effect. Note that this
explanation of OFQ/N⬘s anti-opioid action differs from
an analogous study of the anti-opioid peptide, CCK,
which was found to specifically attenuate the opioid
activation of OFF cells without affecting ON cell firing
(Heinricher et al., 2001).
A subsequent study by Pan et al. (2000) replicated the
inhibition of ON and OFF cells by OFQ/N, as measured
intracellularly with whole-cell patch clamp, and showed
that OFQ/N blocked the electrophysiological and analgesic effects of ␮ and ␬ agonists. They performed an
intriguing additional experiment, however. ON cells
have been implicated in opioid withdrawal-associated
hyperalgesia (Bederson et al., 1990), and these investigators demonstrated that during naloxone-precipitated
acute opioid withdrawal, OFQ/N iontophoretically applied into the RVM produced an anti-hyperalgesic (i.e.,
analgesic) action (Pan et al., 2000). Thus, under different behavioral conditions, two opposing effects could be
elicited from OFQ/N: anti-analgesia and anti-hyperalgesia. It is quite unlikely that those observing supraspinal
OFQ/N analgesia were testing opioid-dependent animals, of course, but the authors suggest that other factors may alter behavioral states in an analogous way,
leading to qualitatively different OFQ/N effects (Pan et
al., 2000).
OFQ/N effects on RVM neurons are not a full explanation, especially since OFQ/N injected into the RVM
did not block systemic morphine analgesia in the radiant
heat tail-withdrawal assay (Heinricher et al., 1997). The
RVM is a sufficient, but not necessary, substrate for
opioid analgesia since lesions of this structure do not
eliminate systemic opioid analgesia (Proudfit, 1980). It
is interesting, therefore, that Morgan and colleagues
(1997) demonstrated that OFQ/N microinjected into the
PAG along with either morphine or kainic acid was able
to reverse both their analgesic actions. OFQ/N inhibits
virtually all neurons studied in a PAG tissue slice
(Vaughan et al., 1997), and thus these authors arrived at
a conclusion mirroring that of the RVM workers: OFQ/N
produced anti-analgesic effects by inhibiting analgesiaproducing neurons (presumably PAG output neurons
projecting to the RVM) downstream from the opioidsensitive neurons (Morgan et al., 1997). Consistent with
these results is a recent electrophysiological study showing that microinjection of OFQ/N into the PAG increased
C-fiber evoked responses and facilitated postdischarge
in spinal wide dynamic range neurons (Yang et al.,
2001).
Thus, there may really be no paradox at all between
the opposing actions of OFQ/N in the spinal versus supraspinal compartments. The analgesic actions of
OFQ/N in the spinal cord can be attributed to the direct
inhibition of nociceptive transmission, actions similar to
those of the classical opioids. By contrast, the supraspinal circuitry, at least in the RVM and PAG, appears to
be set up in such a way that opioid analgesia requires
disinhibition. Any compound, like OFQ/N, producing
ubiquitous cellular inhibition will thus act to oppose
opioid analgesia. Ultimately, then, the differential behavioral actions between supraspinal OFQ/N and classical opioids can be attributed solely to the fact that
their respective receptors are located on functionally
different groups of neurons.
The explanation detailed above has been challenged
by the very recent findings of Rady and colleagues
(2001), who argue that OFQ/N⬘s anti-analgesic actions
can be explained by the ability of supraspinally injected
OFQ/N to activate a descending anti-analgesic system
that releases PGE2 in the spinal cord. Their study was
MOGIL AND PASTERNAK
the first to demonstrate that supraspinal OFQ/N also
blocks spinal morphine analgesia, which cannot be easily explained by a mode of action confined to the supraspinal compartment. Furthermore, they showed that
the cyclooxygenase inhibitor, indomethacin, fully reversed this anti-analgesic action of OFQ/N, and that
PGE2 could mimic OFQ/N⬘s anti-analgesia (Rady et al.,
2001). Both OFQ/N⬘s and PGE2’s anti-analgesia could be
reversed by PGD2, and evidence was provided implicating spinal EP1 receptors in the effect. Although the
involvement of PGE2 and EP1 receptors and the blockade by PGD2 is reminiscent of Ito’s laboratory’s work
regarding spinal OFQ/N allodynia (Minami et al., 1997)
(see Section VIII.D.5.), it appears that different mechanisms are responsible for supraspinal anti-analgesia,
since it is very unlikely that supraspinal OFQ/N releases spinal OFQ/N (Rady et al., 2001).
IX. Effects of Related Peptides on Pain
OFQ/N is not the only bioactive peptide derived from
the ppOFQ/N gene. We now turn to a consideration of
the actions of the other major maturation products, nocistatin and OFQ/NocII, in hopes that they may shed
light on the elusive role of this system in nociceptive
modulation.
A. Nocistatin
Nocistatin originally was reported to reverse, in a
dose-dependent manner, the allodynia and hyperalgesia
produced by low doses of spinal OFQ/N or PGE2 (OkudaAshitaka et al., 1998). An endogenous role for the peptide was suggested by the further demonstration that
the inverted U-shaped dose-response curve for spinal
OFQ/N allodynia could be shifted to the left by 500-fold
with nocistatin antibody. Although only the data obtained with bovine nocistatin were presented, related
mouse, rat, and human sequences were also reported to
inhibit OFQ/N and PGE2 allodynia (Minami et al., 1998;
Okuda-Ashitaka et al., 1998). Just as OFQ/N appears to
functionally antagonize the analgesic actions of opioid
peptides, nocistatin appeared to functionally antagonize
the actions of OFQ/N. This conclusion is further supported the inability of nocistatin to compete binding to
the NOP1 receptor.
Further research reveals a more complex interaction
between nocistatin and OFQ/N. Supraspinal nocistatin
dose-dependently blocked supraspinal OFQ/N⬘s blockade of morphine analgesia, despite having no effect on
basal radiant heat tail-withdrawal latencies or morphine analgesia itself, further supporting the concept
that nocistatin is a functional OFQ/N antagonist (Zhao
et al., 1999). However, nocistatin does not oppose all of
OFQ/N⬘s effects. Although spinal nocistatin reversed
the hyperalgesic action of spinal OFQ/N on the tonic
phase of the 1% formalin test in mice, it was unable to
reverse an analgesic action of OFQ/N on the 2% formalin
403
test in mice (Nakano et al., 2000) and 5% formalin test in
rats (Yamamoto and Sakashita, 1999a). Thus, in these
assays nocistatin appeared to be ineffective against analgesic actions of OFQ/N.
Nocistatin interacted with OFQ/N in altering the
flexor reflex of spinalized rats in a highly complex manner, including an enhancement of the facilitatory effect
of low dose OFQ/N (Xu et al., 1999b). Although nocistatin blocks OFQ/N⬘s inhibition of potassium-evoked glutamate release from rat brain slices (Nicol et al., 1998),
a number of other non-nociceptive actions of OFQ/N are
not reversed by nocistatin (Okuda-Ashitaka and Ito,
2000).
The effects of nocistatin on nociceptive processing also
are uncertain. Spinal or supraspinal nocistatin alone
has no effect on nociceptive thresholds on the hot-plate,
radiant heat tail-withdrawal, or paw-pressure tests
(Okuda-Ashitaka et al., 1998; Nakagawa et al., 1999;
Zeilhofer et al., 2000; Zhao et al., 1999). Although Nakano et al. (2000) demonstrate non-naloxone-reversible
analgesic effects of low doses of spinal nocistatin on both
phases of the formalin test, higher doses are ineffective
against the tonic phase (Yamamoto and Sakashita,
1999a). Also, Zeilhofer and colleagues (2000) demonstrate enhanced formalin responding by low doses of
spinal nocistatin. Supraspinally administered nocistatin
is anti-hyperalgesic when administered alone (Nakagawa et al., 1999).
Okuda-Ashitaka and Ito (2000) summarize this literature by suggesting that nocistatin antagonizes OFQ/N
at low doses but not at higher doses. Given that the
effects of OFQ/N itself may also depend on dose (see
Section VIII.D.5.), they further propose that different
receptors may be responsible. To date, of course, only
one receptor (NOP1) has been found for OFQ/N, although OFQ/N receptor heterogeneity has been suggested (see above), and none has been identified for
nocistatin. Recent studies reveal that nocistatin attenuates transmitter release from inhibitory GABAergic and
glycinergic interneurons in the dorsal horn whereas
OFQ/N blocks excitatory glutamatergic synaptic transmission (Zeilhofer et al., 2000), which provides additional insights into the actions of these peptides.
Nocistatin has been detected in human brain tissue,
and in the cerebrospinal fluid of two chronic pain patients, one with chronic low backache and one with knee
pain (Lee et al., 1999). The authors suggest, somewhat
speculatively, that chronic pain may induce production
of nocistatin in spinal cord; this possibility should be
investigated in a controlled manner.
B. Orphanin FQ/Nociceptin 2
OFQ/NocII is a heptadecapeptide immediately downstream from OFQ/N. Only three studies have examined
its effects on nociception, but the results are not consistent. Rossi and colleagues (1998a) demonstrated dosedependent analgesia after both spinal and supraspinal
404
injection. Like the supraspinal analgesia obtained by
these investigators using OFQ/N, OFQ/NocII analgesia
was greatly enhanced by pretreatment with the ␴1 receptor ligand, haloperidol. Although supraspinal OFQ/
NocII analgesia was naloxone reversible, spinal OFQ/
NocII analgesia was not, suggesting the possibility of
separate OFQ/NocII receptors in the spinal cord or different types of neuronal circuits (Rossi et al., 1998a).
Another group using similar doses and mice was unable
to demonstrate supraspinal OFQ/NocII analgesia on the
hot-plate, tail-flick, or acetic acid abdominal constriction
tests (Florin et al., 1999). In fact, the authors reported a
significant decrease in hindpaw licking latencies on the
hot plate at several doses. The authors did not interpret
this decrease as reflecting hyperalgesia, although, since
rearing and jumping latencies were not similarly affected (Florin et al., 1999). The related peptide, NocIII,
corresponding to OFQ/NocII with an additional three
arginines at the carboxyl terminus, did not appear to
have any biological activity whatsoever. Finally, OkudaAshitaka and colleagues (1998) reported that bPNP-4,
which is identical to OFQ/NocII, possessed nocistatinlike anti-OFQ/N properties when injected spinally. No
spinal analgesia was observed from bPNP-4, although
much lower doses were used.
Reconciling these studies is not easy. Several issues
may prove important. First, differences in the assay
might change the apparent levels of analgesia. Second,
the use of haloperidol reportedly increases the analgesic
activity of OFQ/NocII significantly; it would have been
interesting if the Florin group had examined OFQ/NocII
in conjunction with haloperidol. Finally, it should be
noted that OFQ/NocII is not an easy compound to work
with. It is quite hydrophobic, difficult to keep in solution
and “sticky”; given time, it will come out of solution and
deposit along the walls of containers. Thus, particular
care must be taken to ensure that drug is not lost along
the walls of tubes, etc., thereby lowering the effective
dose injected.
Finally, the sequence of ppOFQ/N raises the possibility of another, longer peptide containing the OFQ/NocII
sequence at its amino terminus. This larger peptide,
mouse ppOFQ/N160 –187, is present within the brain and
is analgesic both spinally and supraspinally; its actions
are reversed by opioid antagonists (Mathis et al., 2001).
The activity of this larger peptide raises the question of
whether it is active by itself or only through the further
processing to OFQ/NocII. The longer peptide also is active in rats (G. C. Rossi, J. Mathis, G. W. Pasternak, and
R. G. Allen, unpublished observations).
X. Involvement of NOP1 in Other Central
Nervous System-Mediated Behaviors
It would be naı¨ve to expect that a peptide with such
broad localization would have biological actions restricted to the modulation of nociception. Although the
lion’s share of the studies has been performed by pain
researchers, OFQ/N and its receptor have been implicated in any number of other phenomena (see Table 5).
Below, we briefly address the role of OFQ/N in several
important behavioral domains featuring mediation by
the CNS.
A. Locomotor Activity and Reward
One of the seminal investigations of OFQ/N reported a
dose-dependent decrease in locomotor activity (i.e., hypolocomotion) when the peptide was given supraspinally
(Reinscheid et al., 1995), as measured by an automated,
photocell-based activity monitor. This effect was significant only at the 10-nmol dose for both horizontal activity (i.e., walking) and vertical activity (i.e., rearing) and
was accompanied by muscular flaccidity, ataxia, and
loss of the righting reflex in two-thirds of the mice
tested. This finding was the first to suggest that OFQ/N
possessed biological activity in vivo and spurred the
investigators to conduct their subsequent nociception
experiments.
The demonstration of changes in locomotor activity
after injection of a compound may have a number of
different interpretations. A decrease in movement, especially when associated with the inability to perform coordinated actions (e.g., ataxia), may indicate that the
dose being administered is too high, exerting nonspecific
effects. On the other hand, a number of psychoactive
drugs can produce alterations in the motivation to move
or, conversely, the motivation to stay still. Thus,
changes in locomotor activity may derive from psychologically important states such as reward/reinforcement,
novelty seeking, and fear/anxiety. Finally, a drug’s alteration of locomotor activity might indicate that it affects neural circuitry (e.g., in the striatum) involved in
the production and regulation of complex motor patterns.
A number of investigators pursued the initial observation of Reinscheid and colleagues (1995). One early
study replicated the hypolocomotion, muscular flaccidity, and ataxia in rats and demonstrated that all of these
signs showed tolerance after repeated injections of 10
nmol of OFQ/N (Devine et al., 1996b; Walker et al., 1998;
Lutfy et al., 2001). In mice, both 1 and 10 nmol of OFQ/N
inhibited locomotion in the open field, as did the highaffinity NOP1 ligand, ac-RYYRIK-NH2 (Noble and
Roques, 1997; Berger et al., 2000). In one study, the
hypolocomotion from 1 nmol lasted only 15 min compared with prolonged effects from 10 nmol; the low-dose
effect could be prolonged, however, by treatment with
inhibitors of aminopeptidase N and endopeptidase
24.15, enzymes that hydrolyze OFQ/N (Noble and
Roques, 1997). The fact that these metabolic inhibitors
were ineffective in altering locomotor activity by themselves allowed the authors to conclude that OFQ/N has
very low tonic release, at least in regions relevant to this
behavior. When microinjected directly into the hip-
MOGIL AND PASTERNAK
pocampus or ventromedial hypothalamus, but not the
nucleus accumbens, high doses of OFQ/N (10 –25 nmol)
significantly decrease locomotor activity (Sandin et al.,
1997; Stratford et al., 1997). Finally, in accordance with
these findings, the repeated injection of antisense oligodeoxynucleotides directed against ppOFQ/N mRNA
produced significant hyperlocomotion in rats (Candeletti
and Ferri, 2000).
This field is not entirely without contradictions, however, because Florin and colleagues (1996), using a much
wider dose range (1–10,000 ng) of OFQ/N, observed significant and dose-dependent, although short-lasting, increases in locomotor activity, in both horizontal and
vertical counts. In addition, they demonstrated an increase in exploratory behavior in the hole-board test
produced by supraspinal OFQ/N injection. These increases were not affected by naloxone, but were blocked
by antagonists of both the dopamine D1 and D2 receptors
(Florin et al., 1996). This same group obtained similar
data using OFQ/NocII in separate studies (Florin et al.,
1997, 1999).
The conflicting findings can be reconciled by considering dose. Studies reporting hypolocomotion feature
OFQ/N doses 2- to 20-times higher than those producing
hyperlocomotion, as seen in the studies by Florin et al.
(1996). In general, the lower doses of a compound are
more likely to elicit physiologically relevant effects than
those seen at higher doses. However, it is always possible that effects at higher doses are produced at alternate, lower affinity binding sites. Indeed, Florin and
colleagues (1996) suggested that the depression of locomotion at high OFQ/N doses might be explained by
nonspecific binding of the peptide to ␬ receptors (see
below). The locomotor-inhibiting effects of 10 nmol of
OFQ/N seen in wild-type animals were not seen in the
NOP1 knockout mice, confirming the importance of the
NOP1 receptor in the effect (Nishi et al., 1997; Noda et
al., 1998). However, the authors did not observe an effect
of 1 nmol of OFQ/N in any genotype. The fact that the
knockout mice did not display any evidence of basal
hyperactivity prompted these investigators to conclude
that this system is not a major player in the regulation
of locomotion (Nishi et al., 1997; Noda et al., 1998).
However, changes in locomotor activity may be caused
by the activity of brain reward/reinforcement systems.
Many abused psychostimulants increase the locomotor
activity of animals that are placed in empty home cages
or open fields (Gold et al., 1989). The increased movement is assumed to be reflective of the induction of a
reward state in the animal, which, futilely, seeks a reinforcing object to consume (e.g., food, water, sex partner). The mesolimbic dopamine pathway projecting from
the ventral tegmental area (VTA) to the nucleus accumbens has long been known to play a crucial, although
controversial, role in the neural processing of reward
(Wise and Bozarth, 1987; Berridge and Robinson, 1998),
and classical opioid peptides acting at ␮ and ␦ receptors
405
can potently release dopamine in the nucleus accumbens
(Devine et al., 1993). In contrast, ␬-selective compounds
including dynorphin are aversive when administered
centrally (e.g., Mucha and Herz, 1985) and inhibit dopamine release in the nucleus accumbens (Spanagel et al.,
1992; Devine et al., 1993). Struck by the fact that endorphins and enkephalins stimulate locomotor activity
whereas dynorphin inhibits it (Chaillet et al., 1983),
Murphy and colleagues (1996) reasoned that the reported hypolocomotor actions of OFQ/N may reflect an
inhibition of mesolimbic dopamine release. Indeed, they
demonstrated that intracerebroventricular or VTA-injected OFQ/N produced a dose-dependent inhibition of
dopamine outflow in anesthetized rats as measured by
microdialysis (Murphy et al., 1996; Murphy and Maidment, 1999). This effect was blocked by the GABAA
antagonist, bicuculline, suggesting its mediation by the
same GABAergic interneurons in the VTA that are affected by opioids (Johnson and North, 1992). In subsequent studies in freely moving rats, supraspinal OFQ/N
did not produce similar effects on basal dopamine levels,
but it completely abolished morphine-induced increases
in dopamine release in the (reward-relevant) shell of the
nucleus accumbens but not in the (reward-irrelevant)
caudate nucleus (Di Giannuario et al., 1999; Di Giannuario and Pieretti, 2000).
These microdialysis findings strongly suggested that
OFQ/N might block the rewarding properties of opiates
and other drugs of abuse. Accordingly, OFQ/N (3–10
nmol but not 30 nmol) abolished the acquisition of a
morphine conditioned place preference (CPP) (Murphy
et al., 1999), in which animals are conditioned to associate a drug with environmental cues and subsequently
assessed for their altered preference for that environment. In studies by another laboratory, even lower
OFQ/N doses abolished morphine CPP (Ciccocioppo et
al., 1999, 2000). The effect of OFQ/N was independent of
the peptide’s ability to impair spatial learning, and unrelated to the development of sensitization processes
(Ciccocioppo et al., 2000). Unlike other anti-opioids (e.g.,
naloxone and dynorphin) that are aversive per se,
OFQ/N has no intrinsic motivational effects since it induces neither a CPP nor a conditioned place aversion
when given alone over a wide dose range (Devine et al.,
1996a ; Ciccocioppo et al., 1999, 2000).
However, the generalized conclusion that OFQ/N opposes the rewarding properties of all opioids is countered
by yet another study. Walker and colleagues (1998) observed that OFQ/N failed to affect the intravenous selfadministration of heroin in the rat. Self-administration
is clearly the “gold standard” of reward/reinforcement
paradigms, but recent evidence points to a dissociation
of the opioid mechanisms underlying heroin and morphine analgesia (Rossi et al., 1996a; Brown et al., 1997;
Schuller et al., 1999; Walker et al., 1999).
Given the considerable literature implicating the involvement of opioid and mesolimbic dopamine systems
406
in alcohol addiction (Herz, 1997; Cowen and Lawrence,
1999), it is not surprising that OFQ/N affects the rewarding properties of ethanol. Using rats artificially
selected to prefer alcohol (Marchigian Sardinian line),
daily injections of intracerebroventricular OFQ/N attenuated consumption in a subchronic (7-day) protocol in
which rats were offered 10% ethanol for 2 h per day
(Ciccocioppo et al., 1999). Blood alcohol levels were unaffected by OFQ/N injection, ruling out a pharmacokinetic explanation of the effect. The peptide also blocked
the acquisition of an ethanol CPP in these animals (Ciccocioppo et al., 1999). However, in an acute protocol, a
single intracerebroventricular injection of OFQ/N increased ethanol consumption. These seemingly contradictory findings can be reconciled by assuming that the
rat attempts to compensate for the blockade of reward in
the acute situation by increasing consumption of the
reinforcer, but abandons this futile strategy over the
longer term. In related studies, antisense knockdown of
the NOP1 receptor increased ethanol-induced hyperlocomotion in rats (Pohorecky et al., 1998) and OFQ/N
blocked stress-induced ethanol self-administration, a
model of relapse (Martin-Fardon et al., 2000). Of course,
this latter finding may be better explained by OFQ/N⬘s
well documented anxiolytic actions (Jenck et al., 1997)
(see Section X.B.) than by a blockade of ethanol reward.
In the only two published studies (Lutfy et al., 2001;
Narayanan and Maidment, 1999) of the direct effect of
OFQ/N on psychostimulant (e.g., amphetamine, cocaine)
reward, the peptide inhibits cocaine-induced hyperlocomotion. Contrary to predictions, the sensitization of cocaine-induced locomotor activation was not affected by
OFQ/N, but OFQ/N induced a sensitized response to
subsequent cocaine injection in naı¨ve animals (Narayanan and Maidment, 1999). In the study by Lutfy and
colleagues (2001), the attenuation of cocaine hyperlocomotion by OFQ/N was accompanied by an attenuation of
cocaine-induced dopamine release in the nucleus accumbens. OFQ/N also blocked hyperlocomotion from the direct dopamine receptor agonist, apomorphine, however,
which suggests an additional mechanism of action independent of extracellular dopamine levels. Finally, in
contrast to the blockade of footshock stress-induced ethanol self-administration by OFQ/N, the peptide did not
block stress-induced cocaine self-administration (Martin-Fardon et al., 2000).
B. Anxiety, Fear, and Stress
Of all the behavioral actions of OFQ/N, its apparent
anxiolytic role may be the most fundamental. Furthermore, this particular action may help explain the effects
of OFQ/N on other phenomena. For example, OFQ/N⬘s
effects on locomotor activity, reward, feeding, pain modulation, and tolerance may be secondary to changes in
stress levels, at least to some extent. The peptide and its
receptor are found in a number of CNS loci involved in
emotion and stress regulation, including the amygdala,
septal region, locus coeruleus, PAG, and hypothalamus
(Herman and Cullinan, 1997). A number of standard
behavioral assays reveal the ability of supraspinal
OFQ/N to block fear and anxiety in both rats and mice
(Jenck et al., 1997). For example, on the elevated plus
maze, a test based on the natural aversion of rodents for
open spaces, OFQ/N in subsedating doses produced a
diazepam-like increase in the time spent in the anxietyprovoking “open arms”. Similar findings reminiscent of
the effects of benzodiapines were observed in the lightdark aversion, open field, and operant conflict tests
(Jenck et al., 1997). Like benzodiazepines, the anxiolytic
effects of OFQ/N generally revealed inverted U-shaped
dose-response curves, likely due to increasing sedation
and ataxia at the highest doses tested. Ro 64 – 6198, a
synthetic agonist of the NOP1 receptor, also was anxiolytic in a large number of behavioral assays (Jenck et al.,
2000). Again, in every case the effects were comparable
to conventional benzodiazepines such as alprazolam or
diazepam, except in a test of panic (Griebel et al., 1995;
Jenck et al., 2000). However, the actions of NOP1 agonists are not identical to those of benzodiazepines, as
illustrated by the absence of anticonvulsant properties
in the former (Jenck et al., 2000).
Griebel and colleagues (1999) extended these findings
using the “mouse defense test battery”, a screening test
for anxiolytics that quantifies behaviors associated with
exposure of a mouse to a rat, a natural predatory threat
stimulus (Griebel et al., 1995). Intracerebroventricularly administered OFQ/N produced significant effects
on some, but not all, of the dependent measures of anxiolysis in this test. OFQ/N was effective against “terminal defense” reactions (e.g., defensive attack, escape attempts), seen when stressful stimuli are unavoidable,
but not against cognitive “risk-assessment” reactions
(Griebel et al., 1999). The authors interpreted the pattern of results to suggest that OFQ/N was primarily
involved in situations of particularly high stress.
ppOFQ/N gene knockout mice also have provided intriguing data regarding the role of OFQ/N in stress. In
addition to their high basal plasma corticosterone levels
and high basal anxiety in three standard behavioral
assays (open field, plus maze, and light-dark box), these
mutants were unable to adapt to repeated stressors (Koster et al., 1999). In this paradigm, mice were stressed
by 10 min of forced swimming in 18°C water. A significant stress-induced analgesia was measured in both
wild-types and knockouts, with the latter genotype having significantly, but not profoundly, more stress-induced analgesia than the former. After two more daily
sessions of forced swimming, wild-type mice had completely adapted, showing virtually no stress-induced analgesia. In contrast, the stress-induced analgesia in the
knockout mice was indistinguishable from the phenomenon on the first day. It may be tempting to interpret
such data as reflecting a lack of tolerance to the analgesia rather than a lack of adaptation to the stressor,
MOGIL AND PASTERNAK
especially since NOP1 knockouts have been shown to
display deficits in tolerance (see Section X.C.). However,
stress and/or stress hormone treatment also attenuates
tolerance development (e.g., Holaday et al., 1979; Takahashi et al., 1988), and thus the “high stress” status of
these mutants may indeed be responsible for any paucity in tolerance. The authors further suggested that a
failure to adapt to stress-induced analgesia may be responsible for the significantly higher than normal tailflick latencies exhibited by group-housed (and thus
chronically stressed) male knockout mice (see Section
VIII.F.). To our knowledge, NOP1 receptor knockouts
have only been tested on one behavioral assay of anxiety, the elevated plus maze, and not found to differ from
wild types (Mamiya et al., 1998).
The site(s) mediating the anxiolytic effects of OFQ/N
are not clear. In one recent study (Kyuhou and Gemba,
1999), OFQ/N microinjected into the PAG fully inhibited
vocalization in the guinea pig elicited by electrical stimulation of the anterior cingulate cortex. This phenomenon is a model of the separation call of guinea pigs
isolated from their conspecifics (Berryman, 1976).
C. Tolerance and Dependence
The development of tolerance to and dependence upon
exogenously administered opiates limits the utility of
these agents clinically. Tolerance exists at many levels
within the organism. Many studies investigating the
phenomena have focused on cellular changes, either of
␮-opioid receptors themselves or of signal transduction
elements coupled to these receptors (e.g., see Nestler,
1997). A wide range of other transmitter systems has
been implicated. Many groups have documented the importance of the NMDA receptor/nitric oxide cascade in
tolerance (Pasternak and Inturrisi, 1995), with more
recent work demonstrating the involvement of ␦-opioid
receptors as well (Abdelhamid et al., 1991; Kest et al.,
1996; Zhu et al., 1999). Clearly, tolerance and dependence involve a complex series of events that are all
intertwined.
Others have proposed systems-level theories (Harrison et al., 1998). One such theory of tolerance holds that
with repeated administration of opioids, the release of
anti-opioid peptides in the CNS is increased, counteracting the analgesia produced (tolerance) and contributing
to the production of a withdrawal syndrome (dependence) once the opioid administration ceases (Rothman,
1992). Given the strong evidence that OFQ/N is itself an
anti-opioid peptide, the involvement of this transmitter/
receptor system in the phenomena of tolerance and dependence was predicted very early on (Mogil et al.,
1996b).
The first clear evidence that OFQ/N and its receptor
may indeed play a role in tolerance came from NOP1
knockout mice, which showed a partial reduction of morphine tolerance development (Ueda et al., 1997). After
five daily injections, wild-type and heterozygous mice
407
displayed a profoundly reduced analgesic response to
morphine on the tail-pinch test. Mutants, on the other
hand, displayed equivalent peak levels of analgesia on
day 5 and day 1, although the analgesia was slower to
develop and quicker to dissipate (Ueda et al., 1997).
Subsequent work by the same group described 12-fold
versus 3.3-fold rightward shifts (representing tolerance)
in morphine analgesia dose-response curves on the tailpinch test in wild-type and knockout mice, respectively
and replicated the finding on the radiant heat tail-withdrawal test (Ueda et al., 2000). ppOFQ/N knockout animals have not been tested for their tolerance status,
although their inability to adapt to stress-induced analgesia after repeated stress exposure might be interpreted as an inability to develop tolerance to this analgesic manipulation (Koster et al., 1999) (see Section
X.B.).
Probably the best evidence for a role of OFQ/N and
NOP1 in tolerance is provided by the partial reduction of
morphine tolerance by systemic administration of the
nonpeptidic NOP1 antagonist, J-113397 (Ueda et al.,
2000). Intriguing differences were noted when the effects of the antagonist were evaluated separately by
route of administration and nociceptive assay. On the
tail-pinch test, intracerebroventricular administration
of J-113397 produced a partial blockade of tolerance,
whereas intrathecal administration produced a complete blockade. On the radiant heat tail-withdrawal test,
spinal administration again produced a complete blockade, but supraspinal J-113397 was entirely without effect (Ueda et al., 2000). The authors attribute this dissociation to the involvement of supraspinal mechanisms
in the tail-pinch test versus the predominantly spinal
mediation of the reflexive radiant heat tail-withdrawal
response and suggest that the NOP1 receptors of relevance to tolerance are located in the spinal cord. In
support of this contention, they demonstrated by reverse
transcription-polymerase chain reaction that NOP1 gene
expression in the spinal cord was increased by 50% in
tolerant versus nontolerant mice (Ueda et al., 2000). A
supporting independent finding is that chronic morphine infusion produced up to a 43% increase in 125I[Tyr14]OFQ/N binding in the superficial layers of the
spinal cord dorsal horn (Gouarderes et al., 1999).
It is interesting that spinal NOP1 receptors have been
implicated in tolerance whereas OFQ/N exerts anti-opioid actions supraspinally (e.g., Grisel et al., 1996). There
is evidence implicating supraspinal OFQ/N in tolerance.
For example, tolerance to morphine and electroacupuncture analgesia is partially reduced following intracerebroventricular treatment with OFQ/N antibodies (Tian
et al., 1998). The OFQ/N antibody blocked both chronic
(given 30 min a day for 6 days) and acute (given continuously for 6 h) electroacupuncture analgesic tolerance,
and chronic (5– 60 mg/kg, 3 times a day for 6 days)
morphine analgesic tolerance, but not acute (5 mg/kg
every 2 h for 16 h) morphine analgesic tolerance. The
408
authors had no explanation for the failure of OFQ/N to
affect this latter phenomenon. Further work by this
group (Yuan et al., 1999) showed increases in OFQ/N
immunoreactivity in brain perfusates and also in the
amygdala and PAG of morphine-tolerant rats. The increased production and release of OFQ/N occurred more
slowly but lasted longer than that of the anti-opioid CCK
in analogous experiments, suggesting that the two peptides may play complementary roles in the mediation of
tolerance.
The role of OFQ/N and its receptor in dependence is
less clear. Many investigators have assumed that direct
injection of OFQ/N would be likely to induce withdrawal
symptoms in morphine-tolerant and -dependent mice.
No group has convincingly shown this and several
groups have reported that it does not occur (Tian et al.,
1997b; Kotlinska et al., 2000). However, Malin et al.
(2000) reported recently that supraspinal OFQ/N dose
dependently produced withdrawal symptoms by itself in
nontolerant rats. Most puzzling, although, is the reported ability of both supraspinal OFQ/N and the NOP1
antagonist, J-113397, to attenuate withdrawal symptoms in morphine-dependent animals (Kotlinska et al.,
2000; Ueda et al., 2000). It should be noted that Kotlinska and colleagues (2000) observed a dose-dependent
blockade of naloxone-precipitated “wet-dog shakes” in
rats by OFQ/N, whereas Ueda et al. (2000) reported the
attenuation by J-113397 of five other common withdrawal symptoms (jumping, paw tremor, backward locomotion, sniffing, and defecation) in mice. Consistent
with the notion that activation of the NOP1 receptor
contributes to rather than blocks the expression of withdrawal is the finding of the latter group that these same
withdrawal symptoms are reduced or absent in NOP1
knockout mice (Ueda et al., 2000) (see Section VIII.F.).
D. Learning and Memory
Classical opioids like ␤-endorphin and dynorphin
modulate learning and memory processes. ␤-Endorphin
consistently disrupts memory, whereas dynorphin can
either enhance or disrupt it (Noda et al., 2000). The high
density of NOP1 receptors in the anterior cingulate,
frontal cortex, and hippocampus suggested that OFQ/N
may play a role in these phenomena. Indeed, Sandin and
colleagues (1997) showed the ability of OFQ/N [but not
OFQ/N(1–13); Sandin et al., 1999] microinjected into the
hippocampus to severely impair spatial learning in the
hippocampally dependent Morris water maze task (Morris et al., 1982). OFQ/N (Yu et al., 1997), like dynorphin
(Wagner et al., 1993), also can impair the induction of
the electrophysiological phenomenon thought by many
to underlie the synaptic plasticity associated with learning, long-term potentiation (LTP) (Stevens, 1998).
Both of these findings have subsequently been replicated and extended. The modulatory effect of OFQ/N on
LTP is due to postsynaptic mechanisms, including the
inhibition of dentate gyrus granule cells and inhibition
of NMDA receptor-mediated currents (Yu and Xie,
1998). In addition to spatial learning on the Morris
water maze, OFQ/N impairs: 1) acquisition in the stepdown passive avoidance task (Hiramatsu and Inoue,
1999a,b); 2) working memory (spontaneous alteration)
in the Y-maze test (Hiramatsu and Inoue, 1999a,b); and
3) “latent” learning in the water-finding task of spatial
attention (Noda et al., 2000). Nocistatin has no effect on
the two former tasks but effectively blocks the OFQ/N
impairment (Hiramatsu and Inoue, 1999a). Nocistatin
also blocked the impairment in these tasks produced by
the muscarinic receptor antagonist scopolamine, which
suggests an interaction between NOP1 receptors and
cholinergic mechanisms of memory (Hiramatsu and Inoue, 1999b). The only conflicting data in this literature is
the lack of effect of low OFQ/N doses (1–10 pmol) on
spontaneous alternation in the Y-maze test (Mamiya et
al., 1999).
The remainder of the relevant data derive from transgenic mice. NOP1 mutants displayed facilitated performance on both escape latency and “probe task”-dependent measures in the Morris water maze and the passive
avoidance task (Manabe et al., 1998; Mamiya et al.,
1999). In addition, hippocampal LTP was up-regulated
in these animals (Manabe et al., 1998; Noda et al., 2000).
In another study, latent learning on the water-finding
task was enhanced in the knockouts (Mamiya et al.,
1998). The authors proposed that a dopaminergic mechanism was underlying the genotypic difference since
dopamine receptor agonists can impair performance on
this assay (Ichihara et al., 1993), and mutant mice displayed low dopamine levels in frontal cortex. The lack of
genotype differences on the Y-maze test supported their
contention that OFQ/N is not involved in working memory (Mamiya et al., 1999). Finally, mice lacking the
ppOFQ/N gene did not differ from wild-type animals on
the Morris water maze (Koster et al., 1999).
E. Feeding
Classical opioid peptides have well documented effects
on feeding behavior, with ␮, ␦, and ␬ agonists increasing
food intake in rodents (Glass et al., 1999). Soon after its
isolation, Pomonis and colleagues (1996) showed that
supraspinal OFQ/N (1–10 nmol) increased food intake in
the satiated rat. OFQ/N⬘s effects are short-lasting, surprisingly specific to food intake with neither water intake nor 1% sucrose intake affected, and accompanied by
transient hypolocomotion (e.g., Polidori et al., 2000a,b).
OFQ/N hyperphagia can be blocked by antisense treatment to NOP1 mRNA (Leventhal et al., 1998), competitive NOP1 antagonism (Polidori et al., 2000a), and functional antagonism by nocistatin (Olszewski et al.,
2000b). Surprisingly, naloxone/naltrexone pretreatment
also blocks OFQ/N⬘s effects on food intake (Pomonis et
al., 1996; Leventhal et al., 1998), although this is probably due to classical opioid receptors being involved in
feeding control at a distal site or affecting motivational
409
MOGIL AND PASTERNAK
processes related to food intake (see Section X.A.) (Polidori et al., 2000b). The NOP1 agonist, [Phe1␺(CH2-NH)
Gly2]-nociceptin(1–13)-NH2, also increases food intake
more potently than OFQ/N when injected supraspinally
(Polidori et al., 2000a).
A microinjection study suggested that the sites of action of OFQ/N hyperphagia include the ventromedial
hypothalamus (VMH), a locus of crucial but controversial importance in the regulation of feeding and/or
weight control (see King, 1980), and the shell of the
nucleus accumbens (Stratford et al., 1997). A more recent study (Polidori et al., 2000b) using lower doses was
unable to replicate the VMH finding, but it did implicate
the hypothalamic arcuate nucleus as the most sensitive
site tested. The lack of effect of OFQ/N injected into the
fourth ventricle in this same investigation appeared to
rule out the involvement of brain stem loci. However, in
a study of 18 brain regions, supraspinal OFQ/N elevated
Fos-like immunoreactivity in six relevant loci, including
the brain stem nucleus tractus solitarius, which had the
largest (5-fold) increase (Olszewski et al., 2000a).
Regarding the mechanism of OFQ/N⬘s hyperphagic
effect, Polidori and colleagues (2000b) point to the fact
that OFQ/N, like opioids, exerts an inhibitory action on
arcuate nucleus neurons (Wagner et al., 1998). This
alone could explain its hyperphagic action via the inhibition of the release of POMC peptides like ␣-MSH and
ACTH, which in turn inhibit feeding. It should be noted,
however, that OFQ/N interacts with any number of
other feeding-relevant neurotransmitter systems, including serotonin, glutamate, and GABA (Polidori et al.,
2000b). The hyperphagic effect of OFQ/N does appear to
be entirely independent from that of neuropeptide Y
(Polidori et al., 2000a).
Competitive and noncompetitive OFQ/N antagonists
reduce deprivation-induced food intake (Olszewski et
al., 2000b; Polidori et al., 2000a), which implicates this
system in the physiological mediation of this phenomenon. A strong role for OFQ/N in basal feeding regulation
remains to be demonstrated, and alterations in food
intake of antisense-treated or transgenic animals have
not been reported. Of potential clinical interest is the
abstracted report that rats, provided with a fat-rich diet,
became hyperphagic and obese and displayed significantly higher OFQ/N binding density in the arcuate
nucleus and VMH than rats fed a standard diet (Malek
et al., 1999).
XI. Conclusions and Future Directions
Neuropeptides have become increasingly important in
our understanding of brain function since the early description of the enkephalins. OFQ/N offers another example of a neuropeptide discovered as a ligand for a
previously described receptor. Despite its structural
similarities to opioid peptides, particularly dynorphin A,
OFQ/N does not label opioid receptors. Yet, its actions
are intimately intertwined with those of the opioids. As
noted above, its most robust action remains a functional
reversal of opioid analgesia, but OFQ/N also elicits analgesia and has been implicated in a wide range of
additional actions as well. Much confusion remains despite the extensive literature that has arisen over the
past 6 years since its original isolation. Differences
among laboratories may arise from any number of potential factors, but they illustrate the complexity of the
OFQ/N system. It is possible, if not likely, that the wide
range of divergent results are each reproducible. If so,
the differences among the studies implies that OFQ/N
actions are highly dependent upon additional factors
that may, or may not, be appreciated. This makes their
evaluation difficult indeed.
The future of OFQ/N and NOP1 receptor research
remains unclear. There seems little doubt that OFQ/N
and its receptor(s) are important. They are associated
with many actions and have a number of potential therapeutic utilities. One major question to address in the
future is the potential of ligand and receptor heterogeneity. Are OFQ/N(1–11) or OFQ/N(1–7) or other fragments physiologically important? Is there truly NOP1
receptor heterogeneity and if so, can it be exploited in
the design and synthesis of selective agonists and antagonists? What is the interaction between OFQ/N functioning, genotype, and stress? These are the questions to
answer. Only when we understand these issues is it
likely that we will finally be able to comprehend and
integrate the results described in this review.
Acknowledgments. J.S.M. is supported by U.S. Public Health Service Grants DA11394 and DE12735. G.W.P. is supported by a Senior
Scientist Award (DA00220) and research grants (DA02615,
DA07242, and DA06142) from the National Institute on Drug Abuse.
We thank Dr. Alan Gintzler and Dr. Kabir Lutfy for useful discussions regarding this manuscript.
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